Wafer level sequencing flow cell fabrication

ABSTRACT

A method for forming sequencing flow cells can include providing a semiconductor wafer covered with a dielectric layer, and forming a patterned layer on the dielectric layer. The patterned layer has a differential surface that includes alternating first surface regions and second surface regions. The method can also include attaching a cover wafer to the semiconductor wafer to form a composite wafer structure including a plurality of flow cells. The composite wafer structure can then be singulated to form a plurality of dies. Each die forms a sequencing flow cell. The sequencing flow cell can include a flow channel between a portion of the patterned layer and a portion of the cover wafer, an inlet, and an outlet. Further, the method can include functionalizing the sequencing flow cell to create differential surfaces.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/560,585, filed Sep. 19, 2017, entitled “Wafer Level SequencingFlow Cell Fabrication,” and U.S. Provisional Patent Application No.62/669,890, filed May 10, 2018, entitled “Wafer Level Sequencing FlowCell Fabrication,” both of which are commonly assigned and incorporatedby reference in their entirety herein for all purposes.

FIELD

The present invention relates generally to a biosensor for biological orchemical analysis, and more specifically, to methods of formingsequencing flow cells including packaging at the wafer level.

BACKGROUND

High-throughput analysis of chemical and/or biological species is animportant tool in the fields of diagnostics and therapeutics. Arrays ofattached chemical and/or biological species can be designed to definespecific target sequences, analyze gene expression patterns, identifyspecific allelic variations, determine copy number of DNA sequences, andidentify, on a genome-wide basis, binding sites for proteins (e.g.,transcription factors and other regulatory molecules). In a specificexample, the advent of the human genome project required that improvedmethods for sequencing nucleic acids, such as DNA (deoxyribonucleicacid) and RNA (ribonucleic acid), be developed. Determination of theentire 3,000,000,000 base sequence of the haploid human genome hasprovided a foundation for identifying the genetic basis of numerousdiseases.

High-throughput analysis, such as massively parallel DNA sequencing,often utilize flow cells, which contain arrays of chemicals and/orbiological species available for analysis. Assay flow cells used as partof an overall system for biological assays include, in variousconfigurations, a carrier in which an assay substrate may be provided,where a substantial portion of the assay substrate can be used forbiochemical analysis, since the carrier component of the flow cell isdesigned to provide functionalities that in prior art systems wereperformed by the assay substrate itself. The flow cells may be used inautomated systems and may be generally flat for imaging. Variousconfigurations of the components of the flow cells minimize evaporation,yet allow for precise control of fluid intake and evacuation.

The manufacture and use of many current flow cells designs can becostly, and the flow cell design is often inefficient in the utilizationof functionalized surface area, decreasing the amount of data that canbe obtained using the flow cell.

SUMMARY

Embodiments of the invention provide methods of wafer level chippackaging of a nanoarray flow cell for DNA sequencing applications. Thewafer level packaging can substantially reduce the cost of the flow cellfabrication. In some embodiments, hard differential surfaces are formedon the wafers, which can be selectively functionalized for the DNBloading. Hard surfaces formed in the embodiments described here canwithstand standard semiconductor wafer level fabrication and packagingprocesses without any sophisticated constraints, which can improvefabrication and chip packaging yield.

According to some embodiments, a method for forming sequencing flowcells can include providing a semiconductor wafer covered with adielectric layer, and forming a patterned layer on the dielectric layer.The patterned layer has a differential surface that includes alternatingfirst surface regions and second surface regions. The method can alsoinclude attaching a cover wafer to the semiconductor wafer to form acomposite wafer structure including a plurality of flow cells. Thecomposite wafer structure can then be singulated to form a plurality ofdies. Each die forms a sequencing flow cell. The sequencing flow cellcan include a flow channel between the patterned layer and the coverwafer, an inlet, and an outlet. The sequencing flow cell can include oneor more first surface regions in the patterned layer and one or moresecond surface regions in the patterned layer. Further, the method caninclude functionalizing the sequencing flow cell to create differentialsurfaces.

In some embodiments of the above method, the first surface regions arehydrophilic surfaces and the second surface regions are hydrophobicsurfaces. In some embodiments, the first surface regions hydrophobic aresurfaces and the second surface regions are hydrophilic surfaces. Insome embodiments, in a sequencing flow cell, either the first surfaceregions or the second surface regions are hydrophilic surfacesconfigured for receiving nucleic acid macromolecules for sequencing.

In some embodiments, the method also includes forming a plurality ofthrough holes in the semiconductor wafer before attaching the coverwafer, the plurality of through holes configured as inlets and outletsfor the flow cells.

In some embodiments, the method can also include comprising forminginlets and outlets in the cover wafer before attaching the cover waferto the semiconductor wafer.

In some embodiments, the semiconductor wafer can also include a CMOSlayer underlying the dielectric layer.

In some embodiments, forming a patterned layer can include forming ametal oxide layer overlying the dielectric layer on the semiconductorwafer, and patterning the metal oxide layer into a plurality of metaloxide regions. The metal oxide regions are configured to receive nucleicacid macromolecules.

In some embodiments, forming a patterned layer can include forming ametal oxide layer, forming a silicon oxide layer overlying the metaloxide layer, and patterning the silicon oxide layer. Regions of themetal oxide layer not covered by the silicon oxide layer are configuredto receive a nucleic acid macromolecule.

In some embodiments, the method also includes forming a supportstructure on the semiconductor wafer before attaching the cover wafer tothe semiconductor wafer.

In some embodiments, the method also includes bonding the cover wafer tothe support structure.

In some embodiments, the cover wafer can include a glass wafer.

In some embodiments, the method can also include functionalizing thesequencing flow cell, wherein functionalizing the sequencing flow cellcan include exposing the flow channel to materials supplied through theinlet and outlet.

In some embodiments, singulating the composite wafer structure caninclude separating the composite wafer structure into individual diesusing a wafer cutting process.

According to some embodiments, a method for forming sequencing flowcells can include providing a semiconductor wafer having a dielectriclayer overlying a complementary metal-oxide-semiconductor (CMOS) layer.The CMOS layer can include a photo sensing layer including a pluralityof photodiodes, and an electronic circuit layer coupled to the photosensing layer for processing sensed signals. The method can includeforming a patterned layer on the dielectric layer, the patterned layerhaving alternate metal oxide regions and silicon oxide regions. Themethod can include attaching a glass wafer to the semiconductor wafer toform a composite wafer structure. The glass wafer can include aplurality of holes. The composite wafer structure includes a pluralityof sequencing flow cells. Each sequencing flow cell can include a glasslayer having holes configured as an inlet and an outlet of thesequencing flow cell. Each sequencing flow cell can include multiplemetal oxide regions and silicon oxide regions, and a flow channelbetween the glass layer and the multiple metal oxide regions and siliconoxide regions. The composite wafer structure can be singulated to form aplurality of dies, each of which can include a sequencing flow cell.

In some embodiments of the above method, forming a patterned layer caninclude forming a metal oxide layer overlying the dielectric layer onthe semiconductor wafer, and patterning the metal oxide layer into aplurality of metal oxide regions. The metal oxide regions are configuredto receive nucleic acid macromolecules.

In some embodiments, forming a patterned layer can include forming ametal oxide layer, forming a silicon oxide layer overlying the metaloxide layer, and patterning the silicon oxide layer. Regions of themetal oxide layer not covered by the silicon oxide layer are configuredto receive a nucleic acid macromolecule.

In some embodiments, the method also includes bonding the glass wafer tothe semiconductor wafer. This bonding step can be used with variouscombinations of the steps of the method described herein.

In some embodiments, the method also includes functionalizing thesequencing flow cell, and functionalizing the sequencing flow cell caninclude exposing the sequencing flow cell to materials supplied throughthe inlet and outlet. This functionalization step can be used withvarious combinations of the steps described above.

According to some embodiments, a method for forming sequencing flowcells can include providing a semiconductor wafer covered with adielectric layer, and forming a patterned layer on the dielectric layer.The patterned layer can have alternate metal oxide regions and oxideregions. The method can also include forming a plurality of throughholes through the semiconductor wafer, and attaching a glass wafer tothe semiconductor wafer to form a composite wafer structure. Thecomposite wafer structure can then be singulated to form a plurality ofdies, each die forming a sequencing flow cell. The method can alsoinclude functionalizing the sequencing flow cell. Each sequencing flowcell can include a glass layer, multiple metal oxide regions and oxideregions, and a flow channel between the glass layer and the multiplemetal oxide regions and oxide regions. The metal oxide regions areconfigured to receive nucleic acid macromolecules, and through holes inthe semiconductor wafer are configured as inlet and outlet of thesequencing flow cell.

In some embodiments of the above method, forming a patterned layer caninclude forming a metal oxide layer overlying the dielectric layer onthe semiconductor wafer, and patterning the metal oxide layer into aplurality of metal oxide regions The metal oxide regions are configuredto receive nucleic acid macromolecules.

In some embodiments, forming a patterned layer can include forming ametal oxide layer overlying the dielectric layer on the semiconductorwafer, forming a silicon oxide layer overlying the metal oxide layer,and patterning the silicon oxide layer. Regions of the metal oxide layernot covered by the silicon oxide layer are configured to receive anucleic acid macromolecule.

In some embodiments, the method can also include bonding the glass waferto the semiconductor wafer.

In some embodiments, the method can also include functionalizing thesequencing flow cell, wherein functionalizing the sequencing flow cellcan include exposing the sequencing flow cell to materials suppliedthrough the inlet and outlet to form hydrophilic surface regions andhydrophobic surface regions. This functionalization step can be usedwith various combinations of the steps described above.

According to some embodiments, a method for forming a device structurehaving differential surfaces includes providing a substrate and forminga surface layer having alternating first thin film regions and secondthin film regions on the substrate. The method includes forming a firstcovering layer selectively on the first thin film regions by exposingthe surface layer to a first material. The method also includes form asecond covering layer selectively on the second thin film regions andnot on the first thin film regions, by exposing the surface layer to asecond material. The method further includes selecting the firstmaterial and the second material to adjust hydrophobicity of the firstcovering layer and the second covering layer.

In some embodiments of the above method, the first thin film regionscomprise a metal or metal oxide material, the metal oxide materialincluding one or more of anodized aluminum (Al₂O₃), tantalum oxide(Ta₂O₅), niobium oxide (Nb₂O₅), zirconium oxide (ZrO₂), and titaniumoxide (TiO₂).

In some embodiments, the first material can include phosphonic acid orphosphate.

In some embodiments, the second thin film regions comprise a siliconoxide.

In some embodiments, the second material can include silane.

In some embodiments, forming a first covering layer on the first thinfilm regions can include an annealing process after exposing the surfacelayer to a first material.

In some embodiments, the annealing process can include 5 to 15 minutesin an inert ambient at 70° to 90° C.

In some embodiments, the first covering layer is hydrophilic, and thesecond covering layer are hydrophobic.

In some embodiments, the first covering layer has positive charges, andthe second covering layer has negative charges.

In some embodiments, forming the surface layer can include forming ansilicon oxide layer, forming a metal oxide layer overlying the siliconoxide layer, and patterning the metal oxide layer to remove portions ofthe metal oxide layer to form a plurality of metal oxide regions, and toexpose a plurality of silicon oxide regions. The first thin film regionsinclude the plurality of metal oxide regions, and the second thin filmregions include the plurality of silicon oxide regions.

In some embodiments, forming the surface layer can include forming ametal oxide layer, forming a silicon oxide layer overlying the metaloxide layer, and patterning the silicon oxide layer to remove portionsof the silicon oxide layers to form a plurality of silicon oxideregions, and to expose a plurality of metal oxide regions. The firstthin film regions include the plurality of metal oxide regions, and thesecond thin film regions include the plurality of silicon oxide regions.

In some embodiments, forming the first covering layer can includeexposing a metal oxide region to polyvinylphosphonic acid (PVPA) to forma hydrophilic covering layer.

In some embodiments, forming the first covering layer can includeexposing a metal oxide region to 12-Hydroxy dodecyl phosphate,(OH-DDPO₄) in a SAM (self assembled monolayer) to form a hydrophiliichydrophilic covering layer.

In some embodiments, forming the first covering layer can includeexposing a metal oxide region ammonium salt of hydroxy dodecyl phosphateto form a hydrophobic covering layer.

In some embodiments, forming the first covering layer can includeexposing a metal oxide region to a mixture of 12-Hydroxy dodecylphosphate, (OH-DDPO₄) and of hydroxy dodecyl phosphate to form a firstcovering layer of adjustable hydrophobicity.

In some embodiments, forming the second covering layer can includeexposing a silicon oxide region to a hydrophobic silane to form ahydrophobic covering layer.

In some embodiments, the hydrophobic silane can include fluorinatedAlkyl-Silanes or dialkyl-Silanes.

In some embodiments, forming the second covering layer can includeexposing a silicon oxide region to a hydrophilic silane to form ahydrophilic covering layer.

In some embodiments, the hydrophilic silane can include hydroxyakylterminated silane.

In some embodiments, the substrate can include a bare semiconductorsubstrate.

In some embodiments, the substrate can include a semiconductor substrateincluding CMOS circuitry and backside illumination (BSI) sensors.

In some embodiments, the substrate can include a glass material.

According to some embodiments, a device structure having differentialsurfaces includes a substrate and a surface layer having alternatingfirst thin film regions and second thin film regions on the substrate.The device includes a first covering layer selectively formed on thefirst thin film regions, and a second covering layer selectively on thesecond thin film regions and not on the first thin film regions. Thefirst covering layer and the second covering layer are configured tohave different hydrophobicity.

The foregoing, together with other features and embodiments, will becomemore apparent upon referring to the following specification, claims, andaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a semiconductor wafer 100 at anintermediate stage of manufacture of sequencing flow cells according toembodiments of the invention.

FIGS. 2-7 are cross-sectional views illustrating various stages of waferscale packaging of sequencing flow cells according to an embodiment ofthe invention. The processes described in FIGS. 2-7 are carried out onwafer 100 described in FIG. 1.

FIG. 2 is a cross-sectional view of a wafer structure 200 havingdifferential surface patterning over the wafer structure 100 of FIG. 1according to some embodiments of the invention.

FIG. 3 is a cross-sectional view illustrating a wafer structure 300having a cover structure disposed over the wafer structure 200 of FIG. 2according to some embodiments of the invention.

FIG. 4A is a cross-sectional view illustrating a wafer structure 400with backside packaging on the wafer structure 300 of FIG. 3 accordingto some embodiments of the invention.

FIG. 4B is a top view of the cover wafer of FIG. 4A according to someembodiments of the invention.

FIG. 5 is a cross-sectional view of a plurality of individual flow celldies 500 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention.

FIG. 6 is a cross-sectional view of a plurality of individual flow celldies 600 after a functionalization process is applied to the flow celldies 500 of FIG. 5 at an intermediate stage of manufacture of sequencingflow cells according to embodiments of the invention.

FIG. 7 is a cross-sectional view illustrating a plurality of individualflow cell dies 700 after a sample loading process in the sequencing flowcells 600 of FIG. 6 according to embodiments of the invention.

FIGS. 8-13 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 8-13 canbe carried out on wafer 100 described in FIG. 1.

FIG. 8 is a cross-sectional view of a wafer structure 800 havingdifferential surface patterning over the wafer structure 100 of FIG. 1according to some embodiments of the invention.

FIG. 9 is a cross-sectional view illustrating a wafer structure 900having a cover structure disposed over the wafer structure 800 of FIG. 8according to some embodiments of the invention.

FIG. 10 is a cross-sectional view illustrating a wafer structure 1000with backside packaging on the wafer structure 900 of FIG. 9 accordingto some embodiments of the invention.

FIG. 11 is a cross-sectional view of a plurality of individual flow celldies 1100 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention. The singulation process illustrated in FIG. 10 is similar tothe process described above in connection with FIG. 5.

FIG. 12 is a cross-sectional view of a plurality of individual flow celldies 1200 after a functionalization process is applied to the flow celldies 1100 of FIG. 11 at an intermediate stage of manufacture ofsequencing flow cells according to embodiments of the invention.

FIG. 13 is a cross-sectional view illustrating a plurality of individualflow cell dies 700 after a sample loading process in the sequencing flowcells 1200 of FIG. 12 according to embodiments of the invention. Thesample loading process illustrated in FIG. 13 is similar to the processdescribed above in connection with FIG. 7.

FIGS. 14-19 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 14-19 aresimilar to the processes of FIGS. 2-7 carried out on wafer 100 describedin FIG. 1 that already has CMOS circuitry built in. In alternativeembodiments, the processes described in FIGS. 14-19 can be carried outon a bare silicon wafer or other semiconductor wafers without built-incircuitry, as described below.

FIG. 14 is a cross-sectional view of a wafer structure 1400 havingdifferential surface patterning over a bare wafer 301 according to someembodiments of the invention.

FIG. 15 is a cross-sectional view illustrating a wafer structure 1500having through holes formed in the wafer structure 1400 of FIG. 14according to some embodiments of the invention.

FIG. 16 is a cross-sectional view illustrating a wafer structure 1600having a cover structure disposed over the wafer structure 1500 of FIG.15 according to some embodiments of the invention.

FIG. 17 is a cross-sectional view of a plurality of individual flow celldies 1700 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention.

FIG. 18 is a cross-sectional view of a plurality of individual flow celldies 1800 after a functionalization process is applied to the flow celldies 1700 of FIG. 17 according to embodiments of the invention. Thefunctionalization process illustrated in FIG. 18 is similar to theprocess described above in connection with FIG. 6.

FIG. 19 is a cross-sectional view illustrating a plurality of individualflow cell dies 1900 after a sample loading process in the sequencingflow cells 1800 of FIG. 18 according to embodiments of the invention.

FIGS. 20-25 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 20-25 aresimilar to those described above in connection to FIGS. 14-19, which areimplemented over a bare wafer.

FIG. 20 is a cross-sectional view of a wafer structure 2000 havingdifferential surface patterning over a bare wafer according to someembodiments of the invention.

FIG. 21 is a cross-sectional view illustrating a wafer structure 2100having through holes formed in the wafer structure 2000 of FIG. 20according to some embodiments of the invention.

FIG. 22 is a cross-sectional view illustrating a wafer structure 2200having a cover structure disposed over the wafer structure 2100 of FIG.21 according to some embodiments of the invention.

FIG. 23 is a cross-sectional view of a plurality of individual flow celldies 2300 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention.

FIG. 24 is a cross-sectional view of a plurality of individual flow celldies 2400 after a functionalization process is applied to the flow celldies 2300 of FIG. 23 according to embodiments of the invention.

FIG. 25 is a cross-sectional view illustrating a plurality of individualflow cell dies 2500 after a sample loading process in the sequencingflow cells 2400 of FIG. 2 according to embodiments of the invention.

FIG. 26 is a cross-sectional view of a device structure 2600 havingdifferential surface regions according to some embodiments.

FIGS. 27A-27F are cross-sectional views illustrating a method forforming the a device structure of FIG. 26 having differential surfaceregions according to some embodiments.

FIG. 27A shows a thin film layer formed on a substrate.

FIG. 27B shows a second thin film layer formed on a first thin filmlayer.

FIG. 27C shows a patterned mask layer formed on the second thin filmlayer.

FIG. 27D shows a cross-sectional view of a device structure havingalternating thin film regions.

FIG. 27E shows a first covering layer selectively formed on the firstthin film region.

FIG. 27F shows a second covering layer selectively formed on the topsurfaces of the second thin film regions to form a device structurehaving differential surface regions.

FIGS. 28A-28C are cross-sectional views illustrating a method forforming the a device structure having differential surface regionsaccording to alternative embodiments of the invention.

FIG. 28A shows a cross-sectional view of a device structure havingalternating surface regions of first thin film layer and second thinfilm layer.

FIG. 28B shows a first covering layer selectively formed on the firstthin film regions.

FIG. 28C shows a second covering layer selectively formed on the topsurfaces of the second thin film regions to form a device structurehaving differential surface regions.

FIG. 29 is a cross-sectional view of a backside illumination CMOS imagesensor, according to some embodiments

FIG. 30 is a cross-sectional view of a backside illumination CMOS imagesensor with a first passivation layer, according to some embodiments.

FIG. 31 is a cross-sectional view of a backside illumination CMOS imagesensor with a first metal layer, according to some embodiments.

FIG. 32 is a cross-sectional view of a backside illumination CMOS imagesensor with an etched first metal layer, according to some embodiments.

FIG. 33 is a cross-sectional view of a backside illumination CMOS imagesensor with a dielectric layer, according to some embodiments.

FIG. 34 is a cross-sectional view of a backside illumination CMOS imagesensor with a color filter layer, according to some embodiments.

FIG. 35 is a cross-sectional view of a backside illumination CMOS imagesensor with a planarized color filter layer, according to someembodiments.

FIG. 36A is a cross-sectional view of a backside illumination CMOS imagesensor with a second passivation layer, a first material layer, and asecond metal layer, according to some embodiments.

FIG. 36B is a cross-sectional view of a backside illumination CMOS imagesensor with a second passivation layer and a second metal layer,according to some embodiments.

FIG. 37 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor, according to some embodiments.

FIG. 38 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor and microlenses, according to someembodiments.

FIG. 39 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor, microlenses and a third metal layer,according to some embodiments.

FIG. 40 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor, microlenses, a third metal layer, and aplanarization layer, according to some embodiments.

FIG. 41 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor, according to some embodiments.

FIG. 42A is a top view of a two-channel color filter that may be used ina biosensor, according to some embodiments.

FIG. 42B is a top view of a four-channel color filter that may be usedin a biosensor, according to some embodiments.

FIGS. 43A-43C are photographic images showing signals from DNBs atnumerous spots on the array in a BSI CMOS chip at various stages of amultiple step sequencing according to some embodiments.

FIG. 44 is a cross-sectional view of a backside illumination CMOS imagesensor with the mask removed, according to some embodiments.

FIG. 45 is a cross-sectional view of a backside illumination CMOS imagesensor with a first coating selectively applied due to differentialsurfaces, according to some embodiments.

FIG. 46 is a cross-sectional view of a backside illumination CMOS imagesensor with a second coating selectively applied due to differentialsurfaces, according to some embodiments.

FIG. 47 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor with macromolecules, according to someembodiments.

DETAILED DESCRIPTION

Embodiments of the invention provide methods of wafer level chippackaging of a flow cell for DNA sequencing applications. The flow cellcan include one or more nucleic acid arrays comprising template nucleicacids for sequencing. In one approach the arrays are DNA nanoball (DNB)nanoarrays. In another approach the arrays comprise clusters of templatenucleic acids, each cluster comprising amplicons of a single templatemolecule.

The wafer level packaging according to an aspect of the invention cansubstantially reduce the cost of the flow cell fabrication. In someembodiments, hard differential surfaces are etched on the wafers, whichcan be selectively functionalized for the DNB loading. Hard surfacesformed in the embodiments described here can withstand standardsemiconductor wafer level fabrication and packaging processes withoutany sophisticated constraints, which can improve fabrication and chippackaging yield.

I. Wafer Level Fabrication of Flow Cell on CMOS Wafer

FIG. 1 is a cross-sectional view of a semiconductor wafer 100 at anintermediate stage of manufacture of sequencing flow cells according toembodiments of the invention. In the manufacturing ofsemiconductor-based sequencing cells, a wafer can have thousands ofdies, and each die represents a portion of the wafer that will befabricated into a sequencing chip including an array of multiple cells,for example, hundreds of cells or more. For simplicity, FIG. 1 onlyshows regions 11 and 12 in semiconductor wafer 100, which are designedfor two flow cells in two separate dies, and each region is shown tohave only two cell areas, which are illustrated in FIG. 1. Region 11includes cell areas 151-1 and 151-2, and region 12 includes cell areas152-1 and 152 2.

As shown in FIG. 1, semiconductor wafer 100 includes CMOS image sensorlayer 10, CMOS processing circuitry layer 20, and stacking layer 30. Ina stacked technology, CMOS image sensor layer 10 and CMOS processingcircuitry layer 20 can be fabricated separately and then joined togetherin a 3-D stacked device with a stacking interface layer 30.

CMOS image sensor layer 10 includes light sensing components 112, e.g.,photodiodes, formed in a semiconductor 110. Semiconductor layer 110 maybe made of any suitable material, such as, for example, silicon, III-Vgroup on silicon, graphene-on-silicon, silicon-on-insulator,combinations thereof, and the like. Although described herein withrespect to photodiodes 110, it is contemplated that any suitable lightsensing component may be used. The photodiodes 110 may be configured toconvert measured light into current. Photodiodes 110 may include thesource and drain of an MOS transistor (not shown) that may transfer thecurrent to other components, such as other MOS transistors. The othercomponents may include a reset transistor, a current source follower ora row selector for transforming the current into digital signals, andthe like. Although described as being dielectric, it is contemplatedthat the dielectric layer may include any suitable electricallyinsulating material.

CMOS image sensor layer 10 also includes metal wirings 105 formed in adielectric layer 104. The metal wirings 115 may include interconnectionsfor integrated circuit materials and external connections.

CMOS processing circuitry layer 20 is shown as a silicon substrate layer101 for simplicity. However, it is understood that CMOS processingcircuitry layer 20 can include CMOS circuits needed for the sequencingoperation. For example, CMOS processing circuitry layer 20 can includecircuitry for image process, signal processing, and control functionsfor sequencing operation, and external communication.

As shown in FIG. 1, CMOS image sensor layer 10 is configured forbackside illumination (BSI). CMOS image sensor layer 10 and CMOSprocessing circuitry layer 20 can be fabricated separately and thenjoined together in a 3-D stacked device with a stacking layer 30.Stacking layer 30 can include a dielectric layer 102 and vias 103 formedin dielectric layer 102. Vias 103 are used for connecting CMOS imagesensor layer 10 and CMOS processing circuitry layer 20.

FIG. 1 also shows a passivation layer 121 overlying CMOS image sensorlayer 10. Passivation layer 121 may be deposited by conventionalsemiconductor processing techniques (e.g., low temperature plasmachemical vapor deposition, PECVD, sputtering, ALD, spin coating,dipping, etc.) on the substrate layer 110 and the photodiodes 112. Thepassivation layer 121 may include any suitable protective material. Forexample, the passivation layer 121 may include materials such as siliconnitride, silicon oxide, other dielectric material, or combinationsthereof, and the like. The passivation layer 121 may act as an etch stopfor later etching steps, as described further herein. The passivationlayer 121 may alternatively or additionally act to protect the activedevice (i.e., the backside illumination CMOS sensor). The passivationlayer 121 may alternatively or additionally act to protect photodiodes112 from wear caused by frequent use. The passivation layer 121 may betransparent.

Discrete areas, sometimes called “spots” or wells (not shown), at whichanalyte molecules may be localized or immobilized may be formed over orin the first passivation layer 121. Chemical or biological samples maybe placed on or over the discrete areas for analysis. In general, forDNA sequencing, the biological samples comprise a DNA sequencinglibrary. DNBs or other members of a DNA sequencing library, or a clonalpopulation thereof, are localized in the discrete areas.

In some embodiments, CMOS image sensor layer 10 may be adapted fordetecting an optical signal (e.g., fluorescent or chemiluminescentemission) from a corresponding array of biomolecules, where individualbiomolecules may be positioned over (e.g., in spots or wells) one ormore photodiodes such that the one or more photodiodes receive lightfrom the biomolecule. As used herein chemiluminescence includesbioluminescence, such as bioluminescence produced by luciferasereporters.

FIGS. 2-7 are cross-sectional views illustrating various stages of waferscale packaging of sequencing flow cells according to an embodiment ofthe invention. The processes described in FIGS. 2-7 are carried out onwafer 100 described in FIG. 1.

FIG. 2 is a cross-sectional view of a wafer structure 200 havingdifferential surface patterning over the wafer structure 100 of FIG. 1according to some embodiments of the invention. FIG. 2 shows alternatelyexposed regions of a first material 123 and a second material 125 areformed overlying dielectric layer 121 of wafer 100 illustrated inFIG. 1. In the embodiment of FIG. 2, a metal-containing layer 123 may bedeposited by conventional semiconductor processing techniques on thepassivation layer 121 (e.g., by sputtering, e-beam evaporation, thermalevaporation, ALD, etc.). Metal-containing Layer 123 may include anysuitable metal or metal oxide material. For example, the layer 123 mayinclude materials such as tungsten, titanium, titanium nitride, silver,tantalum, tantalum oxide, hafnium, chromium, platinum, tungsten,aluminum, gold, copper, combinations or alloys thereof, and the like.Layer 123 may be opaque to incident light and/or, when present, toexcitation light.

In FIG. 2, regions of layer 125 may be formed by depositing a dielectricmaterial and patterned using a photolithography and etching process. Thedielectric material 125 may include any suitable protective material.For example, the dielectric layer 121 may include materials such assilicon nitride, silicon oxide, other electrically insulating material,or combinations thereof, and the like. Dielectric layer 125 may bedeposited by conventional semiconductor processing techniques (e.g., lowtemperature plasma chemical vapor deposition, PECVD, sputtering, ALD,spin coating, dipping, etc.) over the metal-containing layer 123.

Next, the deposited dielectric layer 121 may be patterned using aconventional photolithography and etching process. The process includesforming a patterned mask on the deposited dielectric layer 121, etchingthe deposited dielectric layer 121, and removing the patterned mask.After the photolithography and etching process, regions ofmetal-containing layer 123 not covered by dielectric layer 125 areexposed. These exposed areas of metal-containing layer 123 may form aspot or well into which biological or chemical samples may be placed, asdescribed further herein.

FIG. 3 is a cross-sectional view illustrating a wafer structure 300having a cover structure disposed over the wafer structure 200 of FIG. 2according to some embodiments of the invention. The cover structure 130can be supported by support structures or spacers 132 disposed over thedielectric layer 125. In some embodiments, cover structure 130 can be aglass wafer having the same dimension as the wafer structure 200 of FIG.2. Cover structure 130 can also be any suitable substrate such as glassmaterials, plastic materials, silica, semiconductor, etc. Coverstructure 130 can be prefabricated with one or more inlets 134 and oneor more outlets 135 for each chip area 151 and 152.

Cover structure 130 can be bonded to the wafer structure 200 of FIG. 2using Support structures or spacers 132. Support structures 132 can helpto define assay regions on the assay substrate. Support structures 132can be made of a suitable dielectric insulating material. In someembodiments, the cover structure may have a thickness less than about300 microns such that said coverslip can accommodate high numericaperture optics with minimal distortion as a viewing window for saidassay regions. The cover structure can be positioned on the spacers soas to support said coverslip with minimal warping and to form one ormore flow channels.

The flow cell components can be directly connected via the use of anadhesive. The adhesive is preferably introduced to a surface thatprovides optimal adhesion between the various flow cell components. Theadhesive may be a solid, such as a tape, or may be an adhesive appliedas a liquid or gel that can subsequently be dried or cured into a solidform. The solid adhesive may provide height to the flow channels byvirtue of its thickness. A liquid or gel can also contain solid orsemi-solid particles of a specific size (e.g., glass or plastic beads)that will remain a particular thickness when the liquid or gel adhesivedries, thus defining the height of the flow channels. In these cases,the adhesive material can form the support structure.

FIG. 4A is a cross-sectional view illustrating a wafer structure 400with backside packaging on the wafer structure 300 of FIG. 3 accordingto some embodiments of the invention. FIG. 4A illustrates a backsidepackaging process at an intermediate stage of manufacture of sequencingflow cells according to embodiments of the invention. Wafer levelpackaging can include TSV (through-silicon via)/RDL (re-distributionlayer) passive interposer. The interposer can support chips on itsbottom-side as well as on its top-side for 3-D integrated circuitintegration. As shown in FIG. 7, through-silicon vias 143 can be formedin silicon wafer 101, which includes CMOS circuits as described above inconnection with FIG. 1. Further, metal routing layer 141 and bondingpads can be formed on the backside contacts to allow communication withexternal circuits and systems.

FIG. 4B is a top view of the cover wafer of FIG. 4A according to someembodiments of the invention. As shown in FIG. 4B, cover wafer 130includes multiple premade holes, which will form the inlets and outletsof the flow cells. As shown in a magnified view of an area 11 designatedas a die for a flow cell, there is an inlet hole 134 and an outlet hole135.

FIG. 5 is a cross-sectional view of a plurality of individual flow celldies 500 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention. The wafer structure 400 in FIG. 4 can include hundreds orthousands of flow cell structures. In between the flow cell structure, athin non-functional spacing, also known as a scribe line, is reserved,where a dicing saw can cut the wafer without damaging the structures andcircuits. The width of the scribe can be very small, typically around100 μm. A very thin and accurate saw is therefore needed to cut thewafer into pieces. The dicing can be performed with a water-cooledcircular saw with diamond-tipped teeth. In FIG. 5, the wafer structurein FIG. 4 is cut or diced into multiple dies or chips in a singulationprocess. Each individual die can contain a single flow cell, such asflow cells 151 and 152 separated by a spacing 150, in FIG. 5.

FIG. 6 is a cross-sectional view of a plurality of individual flow celldies 600 after a functionalization process is applied to the flow celldies 500 of FIG. 5 at an intermediate stage of manufacture of sequencingflow cells according to embodiments of the invention. For example, afirst surface layer 161 and a second surface layer 162, which hasproperties different from the first surface layer, may be selectivelyapplied based on the differential surfaces of the metal-containing layer123 and the dielectric layer 125, respectively. The first and secondsurface layers have different properties, resulting in an array of spotsor wells comprising a bottom surface comprising the first surface layer,separated by areas comprising the second surface layer. In someembodiments, macromolecules (e.g., polynucleotides, DNBs, proteins,etc.) of interest preferentially associate with the first surface layercompared with the second surface layer.

The first surface layer 161 may be formed by exposing the flow channels136 and 137 to a suitable material supplied through the inlet 134 andoutlet 135. The first surface layer 161 may also be selectively appliedto the metal-containing layer 123 based on its surface properties. Forexample, the first surface layer 161 may be of such a material that itmay bond to and/or be attracted to the metal-containing layer 123. Insome embodiments the first surface layer does not bind or adhere to, oris repelled by, the dielectric layer 125. It will be recognized that theterm “surface layer” is not intended to ascribe any particular structureor dimensions.

The first surface layer 161 may include any suitable material thatadheres or binds the metal-containing material 123. In one approach, thefirst surface layer 161 is produced by application of a phosphatecompound that binds metal, including without limitation, inorganicphosphate, phosphoric acid, organic phosphate compounds such ashexmamethyl tetraphosphate, hexamthethylphosphoramide, combinationsthereof, and the like.

In some embodiments, the second surface layer 162 may include a materialthat repels biological or chemical analytes of interest. For example,the second surface layer 162 may include a material that has a negativecharge, thus repelling negatively charged biological or chemicalsamples. In some embodiments, the second surface layer 162 may behydrophobic. Those of ordinary skill in the art will recognize thatcombinations (e.g., pairwise combinations) of metals and the secondsurface layer can be selected and optimized for particular purposes.

In FIG. 6, the second surface layer 162 may be selectively applied tothe dielectric layer 125 based on the surface properties of thedielectric layer. For example, the second surface layer 162 may be ofsuch a material that it may bond to and/or be attracted to thedielectric layer 125, but does not bond to or adhere to the firstsurface layer 161 which covers metal-containing layer 123. The secondsurface layer 162 may be applied by coating or treating the exposedportions of the dielectric layer 125 with a second material. In oneapproach, both the exposed dielectric layer 125 and metal-containinglayer 123 regions covered by the first surface layer 161 are exposed tothe second material, which adheres only on the dielectric layer. Thesecond surface layer 162 may be formed by exposing the flow channels 136and 137 to a suitable material supplied through the inlet 134 and outlet135. In one approach the second surface layer 162 is produced byapplication of silane or a silane compound, including withoutlimitation, 3-aminopropyl-methyldiethoxysilane,aminopropyltrimethoxysilane, 3-aminopropyltri-ethoxysilane, etc.

In some embodiments, the first surface layer 161 may include a materialthat attracts biological or chemical samples. For example, the firstsurface layer 161 may include a material that has a positive charge,thus attracting negatively charged biological or chemical samples. Insome embodiments, the first surface layer 161 may be hydrophilic. Thoseof ordinary skill in the art will recognize that combinations of thefirst surface layer and the second surface layer can be selected andoptimized for particular purposes.

It will be recognized that the term “surface layer” is not intended tolimit the first and second surface layers to any particular method ofapplication or structure. As noted, different properties of the firstand the second surface layers may be selected to differentially retaintarget macromolecule(s), e.g., DNA macromolecules. It will also berecognized that the first and/or second surface layers may befunctionalized such that the functionalized surface has a property thatresults in differential retention of target macromolecule(s). Forillustration, after application of the first and second surface layers aDNA binding molecule (e.g., oligonucleotide) with affinity to the secondsurface layers, but not to the first surface layers, may be applied tocover second surface layer 162. In some embodiments, the second surfacelayer 162 can be a functionalized surface on which a single nucleic acidmolecule is amplified.

Thus, a structure may be created in which a first surface layer ispresent in protruding regions, and a second surface layer is present inrecessed regions between protruding portions. The recessed regions mayform spots or wells into which biological or chemical samples may beplaced. It will be recognized that the term “first surface layer” or“second surface layer” may refer to the material applied to the surfaceas well as the material retained on the surface (e.g., the latter maydiffer from the former by evaporation of a solvent; by a reaction withthe surface material, and the like). It is further noted that thefunctionalization process described here in connection with FIG. 6 canbe performed in other steps of the flow cell formation process. Forexample, the functionalization process can be performed after thedifferential surface patterning process described in connection withFIG. 2, or after the disposition of the cover wafer described inconnection with FIG. 3, or after the backside packaging processdescribed in connection with FIG. 4.

FIG. 7 is a cross-sectional view illustrating a plurality of individualflow cell dies 700 after a sample loading process in the sequencing flowcells 600 of FIG. 6 according to embodiments of the invention.Biological or chemical samples 171 can be introduced into the flowchannels by flowing a liquid through the inlets and outlets of the flowchannels. Embodiments of the invention are not limited to any particularmethod of introduction. In some embodiments, the biological or chemicalsamples 171 may be attracted to or bind to the first surface layer 161,while being repelled by the second surface layer 162.

The biological or chemical samples may include any of a number ofcomponents. For example, a sample may contain nucleic acidmacromolecules (e.g., templates, DNA, RNA, etc.), proteins, and thelike. The sample may be analyzed to determine a gene sequence, DNA-DNAhybridization, single nucleotide polymorphisms, protein interactions,peptide interactions, antigen-antibody interactions, glucose monitoring,cholesterol monitoring, and the like.

As discussed above, in some embodiments the biomolecule is a nucleicacid, such as DNA. See U.S. Pat. Nos. 8,778,849; 8,445,194; 9,671,344;7,910,354; 9,222,132; 6,210,891; 6,828,100; 6,833,246; 6,911,345, andPat. App. Pub. No. 2016/0237488, herein incorporated by reference intheir entireties. Without limitation, the DNA biomolecule may be a DNAnanoball (single stranded concatemer) hybridized to labeled probes(e.g., in DNB sequencing by ligation or cPAL methods) or tocomplementary growing strands (e.g., in DNB sequencing by synthesismethods) or both; or a single DNA molecule (e.g., in single moleculesequencing); or to a clonal population of DNA molecules, such as iscreated in bridge PCR-based sequencing. Thus, reference to “abiomolecule”, “a DNA macromolecule” or “a nucleic acid macromolecule”may encompass more than one molecule (e.g., a DNB associated withmultiple growing complementary strands or a DNA cluster comprisingclonal population of hundreds or thousands of DNA molecules). Exemplarymethods for making DNBs (e.g., DNB libraries) and for making arrays ofdiscrete spaced apart regions separated by inter-regional areas are wellknown in the art. See, for example, U.S. Pat. Nos. 8,133,719; 8,445,196;8,445,197; and 9,650,673, herein incorporated by reference in theirentireties. In some embodiments DNBs or other macromolecules areimmobilized on discrete spaced apart regions, or spots, throughattractive noncovalent interactions (e.g., Van der Waal forces, hydrogenbonding, and ionic interactions). In some embodiments discrete spacedapart regions comprise functional moieties (e.g., amines). In someembodiments discrete spaced apart regions comprise captureoligonucleotides attached thereto, for binding template DNAs (e.g.,DNBs). Generally the discrete spaced apart regions are arranged in arectilinear pattern, however, regular arrays with other arrangements(e.g., concentric circles of regions, spiral patterns, hexagonalpatterns, and the like) may be used.

In some embodiments, the nucleic acid macromolecules may be amplicons ofgenomic DNA fragments or a cDNA library. As used herein, an “amplicon”may be the product of amplification of a nucleic acid molecule,typically a fragment of genomic DNA or a cDNA library. Methods ofamplification include, but are not limited to, rolling circleamplification, as described, for example, in U.S. Pat. No. 8,445,194(herein incorporated by reference in its entirety), or bridge polymerasechain reaction (PCR), as described, for example, in U.S. Pat. No.7,972,820, herein incorporated by reference in its entirety. Theamplification may be performed before the nucleic acid is contacted withthe biosensor, or in situ, as described, for example, in U.S. Pat. No.7,910,354, herein incorporated by reference in its entirety.

For example, a biological sample, such as a DNA macromolecule,oligonucleotide, or nucleotide, associated with a fluorescent orchemiluminescent dye, may be placed above a photodiode 117. In the caseof fluorescence, the dye may be illuminated by excitation light from anexcitation light source. The excitation light may correspond to anysuitable type or intensity of light, including, for example, visiblelight, infrared (IR), ultraviolet (UV), and the like. The excitationlight may also come from any suitable source, such as light emittingdiodes (LEDs), lamps, lasers, combinations thereof, and the like. Whenthe dye is illuminated with excitation light at a certain wavelength,the biological sample may absorb the light, then emit light of adifferent wavelength. For example, the biological sample may absorbexcitation light having a 450 nm wavelength, but emit light with a 550nm wavelength. In other words, fluorescent light of a characteristicwavelength may be emitted when the dye is illuminated by light of acharacteristic different wavelength (i.e., the excitation light source).Because excitation light is used to measure fluorescence, however, itmust be filtered out in order to take accurate measurements at thephotodiode 117.

In the case of chemiluminescence, no excitation light source is neededfor the photodiodes 112 to detect emitted light. Instead, the biologicalsample may emit light due to a chemical or enzymatic reaction that mayoccur between the biological sample and the chemiluminescent dye (orother solution), causing light to be emitted due to breaking or formingchemical bonds (e.g., the action of a luciferase protein on a luciferinsubstrate).

For both fluorescence and chemiluminescence, the photodiodes 117 maydetect the intensity of the emitted light and transform it into anelectronic signal based on the intensity of the light that may beprovided to an external device via metal wiring 105. The external devicemay correlate the electronic signal to a particular wavelength andbrightness, based on the electronic signal.

In some embodiments, the active spot or well on the surface of thebiosensor and the nucleic acid macromolecule may be mutually configuredsuch that each spot binds only one nucleic acid macromolecule. This maybe achieved, for example, by contacting the surface with amplicons thatcorrespond in size to the active spot (e.g., an amplicon having adiameter that is effectively as large or larger than the diameter of theactive spot). See U.S. Pat. No. 8,445,194, herein incorporated byreference in its entirety. Alternatively, the active spot can bechemically adapted to bind a single DNA fragment, which may then beamplified to fill a larger region at and around the original bindingsite.

Some embodiments of the invention may be used to determine differentlabels corresponding to different wavelengths of light. The labels maybe, for example, fluorescent, chemiluminescent or bioluminescent labels.For example, in gene sequencing (or DNA sequencing), embodiments of theinvention may be used to determine the precise order of nucleotide baseswithin a nucleic acid macromolecule (e.g., a strand of DNA). Thenucleotide bases may be labeled with a specific fluorescent label (e.g.,adenine (A), guanine (G), cytosine (C), or thymine (T)). Alternatively,one color, two color, or three color sequencing methods, for example,may be used.

With respect to fluorescence, each of the nucleotide bases may bedetermined in order by successively exciting the nucleic acidmacromolecule with excitation light. The nucleic acid macromolecule mayabsorb the excitation light and transmit an emitted light of a differentwavelength onto a biosensor as described herein. The biosensor maymeasure the wavelength of emitted light and intensity received by thephotodiode. Each nucleotide (e.g., fluorescently labeled nucleotide),when excited by excitation light of a certain wavelength and/orintensity, may emit a certain wavelength of light and/or intensity intothe photodiode, allowing identification of the presence of a particularnucleotide base at a particular position in the nucleic acidmacromolecule. Once that particular nucleotide base has been determined,it may be removed from the nucleic acid macromolecule, such that thenext successive nucleotide base may be determined according to a similarprocess.

A nucleic acid macromolecule may be labeled with one or more differentfluorescent, chemiluminescent, or bioluminescent labels before or afterattaching to the biosensor for any purpose. For example, the nucleicacid macromolecule may be hybridized with a labeled oligonucleotideprobe or amplification primer. Alternatively, the nucleic acidmacromolecule may be hybridized with a non-labeled oligonucleotide,which may then be ligated to a labeled probe, or extended using labelednucleotide analogs. By way of illustration, the labeling may be done forthe purpose of characterizing the nucleic acid macromolecule (forexample, the presence of a single nucleotide polymorphism (SNP)associated with a disease), or for nucleic acid sequencing of all or apart of the nucleic acid macromolecule, as described above. DNAsequencing by probe hybridization is described, for example, in U.S.Pat. No. 8,105,771, herein incorporated by reference in its entirety.Sequencing by anchor probe ligation is described, for example, in U.S.Pat. No. 8,592,150, herein incorporated by reference in its entirety.Sequencing by synthesis is described, for example, in U.S. Pat. No.7,883,869, herein incorporated by reference in its entirety. In general,sequencing by synthesis is a method in which nucleotides are addedsuccessively to a free 3′ hydroxyl group provided by a sequencing primerhybridized to a template sequence, resulting in synthesis of a nucleicacid chain in the 5′ to 3′ direction. In one approach, another exemplarytype of SBS, pyrosequencing techniques may be employed (Ronaghi et al.,1998, Science 281:363.

In some embodiments, the biosensor may be reversibly coupled to a flowcell (not shown). The nucleic acid macromolecule may be attached to thebiosensor by contacting the biosensor with a liquid sample in the flowcell. The flow cell may include one or more flow channels that are influid communication with the reaction sites. In one example, thebiosensor may be fluidicly and electrically coupled to a bioassaysystem. The bioassay system may deliver reagents to the reaction sitesaccording to a predetermined protocol and perform imaging events. Forexample, the bioassay system may direct solutions to flow along thereaction sites. The solution may include four types of nucleotideshaving the same or different fluorescent labels. In some embodiments,the bioassay system may then illuminate the reaction sites using anexcitation light source. The excitation light may have a predeterminedwavelength or wavelengths. The excited fluorescent labels may provideemission signals that may be detected by the photodiodes 117.

A user may prepare for sequencing by contacting a biosensor according todescribed embodiments with nucleic acid amplicons, or with a nucleicacid that is subsequently amplified, such that the nucleic acidmacromolecule binds and is retained by the active spots or wells, andexcess nucleic acid macromolecule may be washed away. The nucleic acidmacromolecules may be contacted beforehand or in situ with a labeledreagent. The biosensor may then be operated as described herein todetermine light emitted on or around nucleic acid macromolecules on thearray. The light may be quantified, or it may be sufficient to determinein a binary fashion which of the nucleic acid macromolecules on thesurface have been labeled with labels that emit at a particularwavelength. Different probes or different nucleic acid analogs may beused concurrently that have labels that emit light at differentwavelengths, for example, to determine different bases at a particularposition in the sequence, or to sequence multiple locations.

Although described herein with respect to a backside illumination CMOSsensor, it is contemplated that embodiments of the invention may besimilarly applied to a frontside illumination CMOS sensor. Further, itis contemplated that embodiments of the invention may similarly apply toany suitable biosensor, such as those biosensors described in U.S.Provisional Pat. App. No. 62/416,813, filed Nov. 3, 2016, which isherein incorporated by reference in its entirety.

II. Alternative Wafer Level Fabrication of Flow Cell on CMOS Wafer

FIGS. 8-13 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 8-13 canbe carried out on wafer 100 described in FIG. 1.

FIG. 8 is a cross-sectional view of a wafer structure 800 havingdifferential surface patterning over the wafer structure 100 of FIG. 1according to some embodiments of the invention. Wafer structure 800,similar to wafer structure 200 of FIG. 2, only shows regions 21 and 22in semiconductor wafer 800, which are designed for two flow cells in twoseparate dies. Wafer structure 800 also has alternately exposed regionsof a first material and a second material that are formed overlyingwafer 100 illustrated in FIG. 1. However, in wafer structure 200, thedielectric regions 125 are formed over the metal-containing regions 123,whereas in wafer structure 800, the metal-containing regions 223 areformed over the dielectric regions 225. In FIG. 8, dielectric regions225 can be formed using the dielectric layer 121 from wafer 200 of waferstructure 200. Dielectric regions 225 also can be formed in anotherdielectric layer deposited over dielectric layer 121. Themetal-containing regions 223 can be formed by a deposition andpatterning process similar to that described above in connection withFIG. 2.

FIG. 9 is a cross-sectional view illustrating a wafer structure 900having a cover structure disposed over the wafer structure 800 of FIG. 8according to some embodiments of the invention. Cover structure 230 andsupport structures 232 can be formed using similar processes asdescribed above in connection with FIG. 3. In FIG. 9, flow channels areformed under the cover structures, and each flow cell can have one ormore inlets and one or more outlets, similar to wafer structure 300.

FIG. 10 is a cross-sectional view illustrating a wafer structure 1000with backside packaging on the wafer structure 900 of FIG. 9 accordingto some embodiments of the invention. The backside packaging in FIG. 10is similar to the backside packaging described above in connection withFIG. 4.

FIG. 11 is a cross-sectional view of a plurality of individual flow celldies 1100 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention. The singulation process illustrated in FIG. 10 is similar tothe process described above in connection with FIG. 5. Aftersingulation, two separate dies 251 and 252 are formed, and divided byscribe line 250.

FIG. 12 is a cross-sectional view of a plurality of individual flow celldies 1200 after a functionalization process is applied to the flow celldies 1100 of FIG. 11 at an intermediate stage of manufacture ofsequencing flow cells according to embodiments of the invention. Thefunctionalization process illustrated in FIG. 12, with two differentsurfaces 261 and 262, is similar to the process described above inconnection with FIG. 6.

FIG. 13 is a cross-sectional view illustrating a plurality of individualflow cell dies 700 after a sample loading process in the sequencing flowcells 1200 of FIG. 12 according to embodiments of the invention. Theloading of samples 271 illustrated in FIG. 13 is similar to the processdescribed above in connection with FIG. 7.

III. Wafer Level Fabrication of Flow Cell on Bare Wafer

FIGS. 14-19 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 14-19 aresimilar to the processes of FIGS. 2-7 carried out on wafer 100 describedin FIG. 1 that already has CMOS circuitry built in. In alternativeembodiments, the processes described in FIGS. 14-19 can be carried outon a bare silicon wafer or other semiconductor wafers without built-incircuitry, as described below.

FIG. 14 is a cross-sectional view of a wafer structure 1400 havingdifferential surface patterning over a bare wafer 301 according to someembodiments of the invention. Wafer structure 1400, similar to waferstructure 200 of FIG. 2, only shows regions 31 and 32, which aredesigned for two flow cells in two separate dies. Wafer structure 1400also has alternately exposed regions of a first material and a secondmaterial that are formed overlying wafer 100 illustrated in FIG. 1. Barewafer 301 is covered with a dielectric layer 321, which can be formed bya deposition process. In wafer structure 1400, the dielectric regions325 are formed over the metal-containing regions 323. The dielectricregions 325 can be formed by a deposition and patterning process similarto that described above in connection with FIG. 2.

FIG. 15 is a cross-sectional view illustrating a wafer structure 1500having through holes formed in the wafer structure 1400 of FIG. 14according to some embodiments of the invention. Through holes 341 areformed to provide inlets and outlets for flow cells described below inconnection with FIG. 16. The through holes can be formed usingconventional patterning and etching processes used in silicon integratedcircuit processing. Each area in wafer structure 1500 designated for aflow cell, such as 351 and 352, can have one or more through holes forforming inlets of the flow cell, and one or more through holes forforming outlets of the flow cell.

FIG. 16 is a cross-sectional view illustrating a wafer structure 1600having a cover structure disposed over the wafer structure 1500 of FIG.15 according to some embodiments of the invention. Cover structure 330and support structures 332 can be formed using similar processes asdescribed above in connection with FIG. 3. A difference is that in FIG.16, the cover structure can be a wafer, such as a glass wafer, withoutany inlet or outlet structures as shown in FIG. 3. In FIG. 16, flowchannels 336 and 337 are formed under the cover structure 330, and eachflow cell can have one or more inlets and one or more outlets formed bythrough holes 341.

FIG. 17 is a cross-sectional view of a plurality of individual flow celldies 1700 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention. The singulation process illustrated in FIG. 17 is similar tothe process described above in connection with FIG. 5. After thesingulation process, flow cell dies or chips 351 and 352 are separatedby a space 350 defined by the scribe lines in the wafer.

FIG. 18 is a cross-sectional view of a plurality of individual flow celldies 1800 after a functionalization process is applied to the flow celldies 1700 of FIG. 17 according to embodiments of the invention. Thefunctionalization process illustrated in FIG. 18 is similar to theprocess described above in connection with FIG. 6. Regions of twodifferent surface layers 361 and 362 are formed.

FIG. 19 is a cross-sectional view illustrating a plurality of individualflow cell dies 1900 after a sample loading process in the sequencingflow cells 1800 of FIG. 18 according to embodiments of the invention.The sample loading process illustrated in FIG. 19 is similar to theprocess described above in connection with FIG. 7. FIG. 19 also shows alight source and camera 380 for providing the illumination and captureof the emission from the flow cells. Alternatively, in applicationswithout illumination, e.g., bioluminescence, block 380 can represent acamera for capture of the emission.

IV. Alternative Wafer Level Fabrication of Flow Cell on Bare Wafer

FIGS. 20-25 are cross-sectional views illustrating various stages ofwafer scale packaging of sequencing flow cells according to anotherembodiment of the invention. The processes described in FIGS. 20-25 aresimilar to those described above in connection to FIGS. 14-19, which areimplemented over a bare wafer.

FIG. 20 is a cross-sectional view of a wafer structure 2000 havingdifferential surface patterning over a bare wafer according to someembodiments of the invention. Wafer structure 2000, similar to waferstructure 1400 of FIG. 14, shows two areas designated for flow cells,such as 41 and 42, and has alternately exposed regions of a firstmaterial and a second material that are formed overlying a wafer.However, in wafer structure 1400, the dielectric regions 325 are formedover the metal-containing regions 323, whereas in wafer structure 2000,the metal-containing regions 423 are formed over the dielectric layer421. The metal-containing regions 423 can be formed by a deposition andpatterning process similar to that described above in connection withFIG. 2.

FIG. 21 is a cross-sectional view illustrating a wafer structure 2100having through holes formed in the wafer structure 2000 of FIG. 20according to some embodiments of the invention. Through holes 441 areformed to provide inlets and outlets for flow cells described below inconnection with FIG. 16. The through holes can be formed usingconventional patterning and etching processes used in silicon integratedcircuit processing. Each area in wafer structure 2100 designated for aflow cell, such as 41 and 42, can have one or more through holes forforming inlets of the flow cell, and one or more through holes forforming outlets of the flow cell.

FIG. 22 is a cross-sectional view illustrating a wafer structure 2200having a cover structure disposed over the wafer structure 2100 of FIG.21 according to some embodiments of the invention. Cover structure 430and support structures 432 can be formed using similar processes asdescribed above in connection with FIG. 3. Similar to the coverstructure 330 in FIG. 16, the cover structure 430 can be a wafer, suchas a glass wafer, without any inlet or outlet structures as shown inFIG. 3. In FIG. 22, flow channels 436 and 437 are formed under the coverstructure 430, and each flow cell can have one or more inlets and one ormore outlets formed by through holes 441.

FIG. 23 is a cross-sectional view of a plurality of individual flow celldies 2300 after a wafer singulation process at an intermediate stage ofmanufacture of sequencing flow cells according to embodiments of theinvention. The singulation process illustrated in FIG. 23 is similar tothe process described above in connection with FIG. 5. After thesingulation process, flow cell dies or chips 451 and 452 are separatedby a space 450 defined by the scribe lines in the wafer.

FIG. 24 is a cross-sectional view of a plurality of individual flow celldies 2400 after a functionalization process is applied to the flow celldies 2300 of FIG. 23 according to embodiments of the invention. Thefunctionalization process illustrated in FIG. 24 is similar to theprocess described above in connection with FIG. 6. In FIG. 24, twodifferent surface layers 461 and 462 are formed.

FIG. 25 is a cross-sectional view illustrating a plurality of individualflow cell dies 2500 after a sample loading process in the sequencingflow cells 2400 of FIG. 2 according to embodiments of the invention. Thesample loading process illustrated in FIG. 25 is similar to the processdescribed above in connection with FIG. 7. In some embodiments, thebiological sample comprises DNA nanoballs (DNBs) 471 which may beattracted to or bind to the first surface layer 161. FIG. 25 also showsa light source and camera 480 for providing the illumination and captureof the emission from the flow cells. Alternatively, in applicationswithout illumination, e.g., bioluminescence, block 480 can represent acamera for capture of the emission.

Although the processes described herein are described with respect to acertain number of steps being performed in a certain order, it iscontemplated that additional steps may be included that are notexplicitly shown and/or described. Further, it is contemplated thatfewer steps than those shown and described may be included withoutdeparting from the scope of the described embodiments (i.e., one or someof the described steps may be optional). In addition, it is contemplatedthat the steps described herein may be performed in a different orderthan that described.

V. Differential Surfaces for Flow Cells

Hydrophobic/hydrophilic interstitial surface with dimension frommicrometers to nanometer has many important biotech applications, suchas forming droplets array inside oil for droplet digital PCR, DNAnanoball array for DNA sequencing, and single cell array for single cellanalysis, etc. In different embodiments, various methods are describedfor forming hydrophobic and hydrophilic interstitial surfaces based onpatterned inorganic surfaces by semiconductor processing techniques.Traditional fabrication methods involve patterning organic hydrophobicpolymer like Teflon, Cytop using special processes and materials, whichare hard to incorporated into standard semiconductor foundry processes.Therefore, such processes are unsuitable for mass production, and theircost can be too high to many applications.

FIG. 26 is a cross-sectional view of a device structure 2600 havingdifferential surface regions according to some embodiments of theinvention. Device structure 2600 includes a substrate 2601. A surfacelayer 2602 including a plurality of first thin film regions 2611 and aplurality of second thin film regions 2621 are disposed on substrate2601. It is further noted that the methods described here are applicableto forming a layer having differential surface regions on any suitablesubstrate 2601, for example, a CMOS device with sensors, a baresemiconductor wafer, a glass substrate, etc. Further details aredescribed below in connection with FIG. 27A, 41, and 44.

A first covering layer 2612 is formed on the top surfaces of the firstthin film regions 2611, and a second covering layer 2622 is formed onthe top surfaces of the second thin film regions 2621. In someembodiments, a differential surface layer is formed by alternatingregions of first covering layer 2612 and regions of second coveringlayer 2622.

According to some embodiments, methods are provided for selecting thefirst material and the second material to adjust the hydrophobicity ofthe first covering layer and the second covering layer to form adifferential hydrophobic/hydrophilic surface.

In some embodiments, differential hydrophobic/hydrophilic surface canhave alternating nonpolar molecular regions that repels water and polarmolecular regions which can form ionic or hydrogen bond with watermolecular. In some embodiments, a method includes first formingalternating regions of inorganic silicon oxide SiO2 and metal oxidematerial including one or more of various metal oxides, such anodizedaluminum (Al₂O₃), tantalum oxide (Ta₂O₅), niobium oxide (Nb₂O₅),zirconium oxide (ZrO₂), and titanium oxide (TiO₂), etc. Thesealternating regions can be formed using standard semiconductor thin filmdeposition and photolithography process on Si or glass substrate, asdescribed further below.

Next, the metal oxide surfaces can be treated to modify the surfaceproperty. One method is to selectively coat it using polyvinylphosphonicacid (PVPA), which is a type of hydrophilic polymer inside the native pHof the phosphonic acid (pH=2), at a temperature in the temperature rangeof 80° C. to 100° C. In a specific embodiment, the treatment can becarried at, for example, 90° C. In some cases, the treatment can berelatively fast, for example, in less than 2 min. This step can befollowed by a dry annealing step intended to support formation ofcovalent bonds. A dry anneal process can be carried at a suitabletemperature, e.g., at 80° C. for about 10 min. This reaction can beselective, i.e., no reaction will take place on the SiO₂ surface.

Another method is to selectively coat the metal oxide regions byself-assembled monolayers (SAMs) based on the adsorption of the alkylphosphate ammonium salts from aqueous solution. SAM formation does notoccur on SiO₂ surfaces under the same conditions. The coated surfacehydrophobicity can be adjusted by the formulation in the aqueous SAMforming solution, and the contact angle of water can range from 50 to110 degrees. In some cases, the contact angle of water can range from 20to 130 degrees. Covalent bonds can be formed during the annealing, andunreacted materials rinsed by DI water.

After the PVPA or phosphate treatment, the substrate can be dried andtreated with a hydrophobic silane such as Fluorinated Alkyl-Silanes,dialkyl-Silanes etc. Alternatively the substrate can be dried andtreated with a hydrophilic silane such as Hydroxyakyl terminated silanesin solution or chemical vapor deposition. These treatment can formstable covalent bonds with the SiO₂ surface and change the surface to behydrophobic or hydrophilic without affecting metal oxide surface'shydrophobicity.

With the highly selective surface treatment of inorganic SiO₂ and metaloxide surfaces with different organic chemicals of differenthydrophobicity, the differential hydrophobic/hydrophilic surface can bemade with well-defined patterns by the semiconductor processes.

FIGS. 27A-27F are cross-sectional views illustrating a method forforming the a device structure of FIG. 26 having differential surfaceregions according to some embodiments of the invention. The method forforming a device structure having differential surfaces includesproviding a substrate, and forming a surface layer having alternatingfirst thin film regions and second thin film regions on the substrate.The surface layer is exposed to a first material to form a firstcovering layer on the first thin film regions, but not on the secondthin film regions. The surface layer is then exposed to a secondmaterial to form a second covering layer on the second thin filmregions, but not on the first thin film regions which are now covered bythe first covering layer. The method includes selecting the firstmaterial and the second material to adjust the hydrophobicity of thefirst covering layer and the second covering layer.

FIG. 27A shows a thin film layer formed on a substrate. In FIG. 27A, athin film layer 2720 is formed on a substrate 2701. Substrate 2701 maybe made of any suitable material, for example, a glass or asemiconductor. A semiconductor substrate can include varioussemiconductor materials, such as silicon, III-V group on silicon,graphene-on-silicon, silicon-on-insulator, combinations thereof, and thelike. Substrate 2701 can be a bare wafer, similar to bare wafer 301 inFIG. 14. Substrate 2701 can also include various device an circuitstructures. For example, substrate 2701 can be similar to semiconductorwafer 100 illustrated in FIG. 1, which can include a CMOS image sensorlayer 10, a CMOS processing circuitry layer 20, and a stacking layer 30.Optionally, substrate 2701 can also include a top passivation layer orinsulating layer (not shown), similar to passivation layer 121 inFIG. 1. The passivation layer may include any suitable protectivematerial. For example, the passivation layer may include materials suchas silicon nitride, silicon oxide, other dielectric materials, orcombinations thereof. The passivation layer may be deposited byconventional semiconductor thin film deposition techniques, e.g.,chemical vapor deposition (CVD), low temperature plasma chemical vapordeposition (LPCVD), plasma-enhanced chemical vapor deposition (PECVD),sputtering, physical vapor deposition (PVD), and atomic layer deposition(ALD), etc.

In FIG. 27A, thin film layer 2720 is formed on substrate 2701. In anembodiment, thin film layer 2720 contains inorganic silicon oxide, e.g.,SiO2. In some embodiments, thin film layer 2720 can include silicon,silicon nitride, metal oxides, etc., or combinations thereof. Thin filmlayer 2720 can also include other materials that can be silanized. Thinfilm layer 2720 can be formed on substrate 2701 using conventionalsemiconductor thin film deposition techniques described above.

FIG. 27B shows a second thin film layer 2710 formed on a first thin filmlayer 2720. In some embodiments, 2710 can include metal oxides ormetals. Suitable metal oxides can include, for example, anodizedaluminum (Al₂O₃), tantalum oxide (Ta₂O₅), niobium oxide (Nb₂O₅),zirconium oxide (ZrO₂), and titanium oxide (TiO₂), etc. Thin film layer2610 can also include metal materials, such as tungsten, titanium,titanium nitride, silver, tantalum, tantalum oxide, hafnium, chromium,platinum, tungsten, aluminum, gold, copper, combinations or alloysthereof, and the like. Thin film layer 2610 can also be formed usingconventional semiconductor thin film deposition techniques describedabove.

FIG. 27C shows a patterned mask layer 2730 formed on thin film layer2710. The mask layer 2730 includes openings exposing regions of thinfilm layer 2710. Mask layer 2730 may be applied according to anysuitable method, such as spin coating, dipping, and/or the like. Masklayer 2730 may also be of any suitable material, such as a photoresist.As shown in FIG. 27C, mask layer 2730 is patterned with the openingsaccording to conventional semiconductor lithography techniques. In someembodiments, mask layer 2630 can be a hard mask, which is a patternedlayer of suitable thin film material having suitable etch selectivity toserve as an etching mask.

After the patterned mask layer 2730 is formed, an etch process can becarried out to remove the exposed portions 2717 in thin film layer 2710.The etch process can be performed according to conventionalsemiconductor process techniques. Subsequently, the patterned mask layer2730 can be removed by conventional semiconductor process techniques.The resulting device structure is illustrated in FIG. 27D.

FIG. 27D shows a cross-sectional view of a device structure havingalternating thin film regions 2711 of thin film layer 2710 and thin filmregions 2721 of thin film layer 2720 according to some embodiments ofthe invention. After the patterning process, a surface layer including aplurality of first thin film regions 2711 and a plurality of second thinfilm regions 2621 are formed on substrate 2701.

FIG. 27E shows a covering layer 2712 selectively formed on thin filmregion 2711. The selective covering layer formation is carried out byexposing the device structure to a suitable first material, such thatcovering layer 2712 is formed on the top surfaces of the thin filmregions 2711, but not on the top surfaces of the thin film regions 2721.Depending on the materials for thin film regions 2711 and 2721, variousmaterials can be used, as described in detail below. After the treatmentof the material, an annealing process can be performed to selectivelyform covering layer 2712 on thin film region 2711. The annealing processcan be carried out at a temperature in a range of 70°−90° C. for 5-15minutes. As an example, a dry anneal process can be carried out at atemperature of 80° C. for 10 minutes. The unreacted material can beremoved by a rinse process in DI (deionized) water.

FIG. 27F shows a second covering layer 2722 selectively formed on thetop surfaces of the second thin film regions 2721. The selectivecovering layer formation is by exposing the device structure to asuitable second material, such that covering layer 2722 is formed on thetop surfaces of the thin film regions 2721, but not on the top surfacesof thin film regions 2711 with covering layer 2712. Depending on thematerials for thin film regions 2711 and 2721, various materials can beused, as described in detail below. After the process with the secondmaterial, FIG. 27F shows a cross-sectional view of a device structure2700, similar device structure 2600 of FIG. 26, having a surface layer2702 with differential surface regions 2712 and 2722 according to someembodiments of the invention. As shown, device structure 2700 includes asubstrate 2701. Surface layer 2702 including a plurality of first thinfilm regions 2711 and a plurality of second thin film regions 2721 aredisposed on substrate 2701.

A first covering layer 2712 is formed on the top surfaces of the firstthin film regions 2711, and second covering layer 2722 is formed on thetop surfaces of the second thin film regions 2721. In this embodiment, adifferential surface layer is formed by alternating regions of firstcovering layer 2712 and regions of second covering layer 2722.

In some embodiments, the differential surface regions can includealternating hydrophilic surfaces and hydrophobic surfaces. In someembodiments, the differential surface regions can include alternatingsurfaces of positive charges and surfaces of negative charges. In thedescription below, thin film regions 2711 are referred to as the firstthin film regions, and thin film regions 2721 are referred to as thesecond thin film regions. Covering layers 2712 are referred to as thefirst covering layers, which are formed by reaction between the firstthin film regions with a first material. Covering layers 2722 arereferred to as the second covering layers, which are formed by reactionbetween the second thin film regions with a second material.

In some embodiments, the first thin film regions can include thin filmsof metal oxides or metals, as described above. Then the metal oxides ormetals can receive a treatment and be exposed to phosphonic acids, suchas PVPA (Polyvinylphosphonic acid). In some embodiments, the treatmentcan be carried out at a temperature ranging from 80° C. to 100° C. for1-3 minutes. For example, the treatment can be carried out at 90 C for 2minutes. This treatment can form a hydrophilic covering layer.

In some embodiments, the metal oxides or metals can receive a treatmentbe exposed to phosphates in a SAM (self-assembled monolayer) process.For example, a SAM process using ammonium salt of hydroxy dodecylphosphate, OH-DDPO₄(NH₄)₂, can form a hydrophobic covering layer with acontact angle of about 110 degrees. In another example, a SAM processusing 12-Hydroxy dodecyl phosphate, (OH-DDPO₄), can form a hydrophiliccovering layer with a contact angle of about 50 degrees. In still otherexamples, a SAM process using a mixture of the different phosphatecompounds can form a covering layer with different hydrophobicity, withcontact angles ranging from 50 to 110 degrees. Further, with suitablecombination of different phosphates, a covering layer with differenthydrophobicity can be formed, with contact angles that can ranges from,for example, 20 to 130 degrees.

After the first covering layers formed on the metal oxides or metalsthin films, a second covering layer can be formed selectively on theregions of the second thin film regions of, e.g., inorganic siliconoxide. For example, a hydrophobic covering layer can be formed bytreating the device in a hydrophobic silane, such as fluorinatedAlkyl-Silanes, dialkyl-Silanes, etc. Alternatively, a hydrophiliccovering layer can be formed by treating the device in a hydrophilicsilane, such as such as Hydroxyakyl terminated silanes, etc. Withappropriate selection of the silane compounds, the second covering layercan be formed only on the second thin film regions of, e.g., inorganicsilicon oxide, and not on the first covering layer already formed on thefirst thin film materials. Besides inorganic silicon oxide, the secondthin film regions can also include materials such as silicon, siliconnitride, metals oxides, or combinations thereof.

In the process described above, the alternating first thin film regions2610 and second thin film layer 2620 are formed in a sequence such thatfirst thin film regions 2610 are formed on second thin film regions2620. In some other embodiments, second thin film regions 2620 can beformed on first thin film regions 2610, as illustrated below in FIGS.28A-28C.

FIGS. 28A-28C are cross-sectional views illustrating a method forforming the a device structure of FIG. 26 having differential surfaceregions according to alternative embodiments of the invention.

FIG. 28A shows a cross-sectional view of a device structure havingalternating surface regions of thin film layer 2810 and thin film layer2820. In FIG. 28A, a surface layer including a plurality of first thinfilm regions 2811 and a plurality of second thin film regions 2821 areformed on substrate 2801.

The device structure in FIG. 28A is similar to that in FIG. 27D, withthe first thin film layer 2810 corresponding to the first thin filmlayer 2710 in FIG. 27D, and the second thin film layer 2820corresponding to the second thin film layer 2720 in FIG. 27D. Onedifference between the structures in FIGS. 27D and 28A is that the firstthin film layer 2810 is below second thin film layer 2820. The devicestructure in FIG. 28A can be formed using a similar process as describedin FIGS. 27A-27C, with the sequence of thin film layer formationreverse.

FIG. 28B shows a covering layer 2812 selectively formed on thin filmregions 2811. The selective covering layer formation is by exposing thedevice structure to a suitable first material, such that covering layer2812 is formed on the top surfaces of the thin film regions 2810, butnot on the top surfaces of the thin film regions 2820. The treatmentprocess and annealing process are similar to those described inconnection with FIG. 27E, with covering layer 2812 corresponding tocovering layer 2712 in FIG. 27E.

FIG. 28C shows a second covering layer 822 selectively formed on the topsurfaces of the second thin film regions 2821. The selective coveringlayer formation is by exposing the device structure to a suitable secondmaterial, such that covering layer 222 is formed on the top surfaces ofthe thin film regions 2821, but not on the top surfaces of thin filmregion 2811 with covering layer 2812. The treatment process is similarto that described in connection with FIG. 27F. After the process withthe second material, FIG. 28C shows a cross-sectional view of a devicestructure 2800, having a surface layer 2802 with differential surfaceregions 2812 and 2822. As shown in FIG. 28C, device structure 2800includes a substrate 2801. Surface layer 2802 including a plurality offirst thin film regions 2811 and a plurality of second thin film regions2821 are disposed on substrate 2801.

A first covering layer 2812 is formed on the top surfaces of the firstthin film regions 2811, and a second covering layer 2822 is formed onthe top surfaces of the second thin film regions 2821. In thisembodiment, a differential surface layer is formed by alternatingregions of first covering layer 2812 and regions of second coveringlayer 2822.

In alternative embodiments, the processes illustrated in FIGS. 27A-27Fand FIGS. 28A-28C can also be modified. For example, the sequence ofsurface layer formation can be reversed with proper selection of thinfilm materials and the compounds for surface treatment. In someembodiments, in FIGS. 27D-27F, covering layers 2722 can be formed onthin film regions 2721 in the structure of FIG. 27D first, and thencovering layer 2712 can be formed on thin film regions 2711. Similarly,in FIGS. 28A-28C, covering layers 2822 can be formed on thin filmregions 2821 in the structure of FIG. 28A first, and then covering layer2812 can be formed on thin film regions 2811.

Although the processes described herein are described with respect to acertain number of steps being performed in a certain order, it iscontemplated that additional steps may be included that are notexplicitly shown and/or described. Further, it is contemplated thatfewer steps than those shown and described may be included withoutdeparting from the scope of the described embodiments (i.e., one or someof the described steps may be optional). In addition, it is contemplatedthat the steps described herein may be performed in a different orderthan that described.

For example, the first and second thin film regions can be either metalor metal oxide, or silicon oxide. Even though in the above example, acovering layer formed by phosphonic acid or phosphate on metal oxides isformed first, followed by a covering layer formed by silane on siliconoxide. In some embodiments, a first covering layer can be formed firstby silane on silicon oxide, and then a second covering layer can beformed by phosphonic acid or phosphate on metal oxides. In someembodiments, the treatment by phosphonic acid or phosphate on metaloxides is followed by an annealing process as described above.

VI. Biosensors for Biological or Chemical Analysis and Methods ofManufacturing the Same

CMOS image sensors find use in electronic imaging devices, includingdigital cameras, medical imaging equipment, radar devices, and the like.Using integrated circuits and a series of photodiodes, CMOS imagesensors can capture light and convert it into electrical signals.

CMOS image sensors are typically implemented on chips. The chips mayhave an amplifier for each pixel. Although the inclusion of manyamplifiers in a chip may result in less area for the capture of light,other components can be integrated onto the chip to direct more lightinto the photodiodes. For example, microlenses may be placed in front ofthe photodiodes to direct light into the photodiodes. To furtherincrease the amount of light that hits the photodiodes, backsideillumination (BSI) can be used. BSI effectively places the photodiodescloser to the light source, instead of under and between the integratedcircuit wiring, reducing destructive interference. BSI CMOS sensors alsohave other advantages. For example, BSI CMOS sensors may have lowoperating voltage, low power consumption, high efficiency, and lownoise.

BSI CMOS image sensors typically have two functional areas: a lightsensing area and an electronic circuit area. The light sensing areaincludes the photodiodes arranged in an array, coupled tometal-oxide-semiconductor (MOS) transistors that detect the lightintensity. The electronic circuit area provides connections between theMOS transistors and external connections, such as to other devices forprocessing the data from the MOS transistors.

In practice, a BSI CMOS image sensor employs filters that divideincident light into bands of light of different wavelengths. The lightis received by the photodiodes on a substrate and transformed intoelectrical signals of different intensity. For example, an incident beammay be divided into red, green, and blue light and received byrespective photodiodes for each color. Each photodiode transforms thedetected light intensity into electrical signals. This is accomplishedby the photodiode accumulating a charge. For example, the higher theintensity of the light, the higher the charge accumulated in thephotodiode. The accumulated charge can then be correlated to a color andbrightness.

In addition to the uses described above, CMOS image sensors may also beused for biological or chemical analysis. For such analysis, abiological or chemical sample may be placed above a photodiode, andlight emitted by the biological or chemical sample may be directed tothe photodiode. The fluorescence or chemiluminescence of the sample canbe detected by the photodiode, and a color and brightness can bedetermined. This color and brightness may be used to identify thebiological or chemical sample.

Embodiments of the invention address the drawbacks associated withprevious approaches by providing an improved biosensor for biological orchemical analysis. According to embodiments of the invention, BSI CMOSimage sensors can be used to effectively analyze and measurefluorescence or chemiluminescence of a sample. This measured value canbe used to help identify a sample. Embodiments of the invention alsoprovide methods of manufacturing an improved biosensor for biological orchemical analysis. As used herein, the term “biosensor” may be used torefer to an apparatus for determining a light emitting substance withinor attached to a biological molecule, particularly a nucleic acidmacromolecule exemplified by DNA and branched or otherwise derivatizednucleic acids. As used herein, the term “nucleic acid macromolecule” mayrefer to, for example, DNB or single strand embodiments.

According to some embodiments of the invention, a biosensor is provided.The biosensor comprises a backside illumination complementarymetal-oxide-semiconductor (CMOS) image sensor. The backside illuminationCMOS image sensor includes an electronic circuit layer and a photosensing layer over the electronic circuit layer. The photo sensing layerincludes a substrate layer and a photodiode in contact with theelectronic circuit layer. A light receiving surface is defined by asurface of the photodiode opposite to the electronic circuit layer. Thebiosensor can also include a color filter material over the photodiode.The biosensor can also include a spot or well above the color filtermaterial that is sized and functionalized to receive a nucleic acidmacromolecule, and to absorb light from the nucleic acid macromoleculeor to pass the light to the light receiving surface from the nucleicacid macromolecule.

A method of manufacture according to some embodiments comprisesproviding a backside illumination complementarymetal-oxide-semiconductor (CMOS) image sensor. Providing the backsideillumination CMOS image sensor includes providing an electronic circuitlayer and providing a photo sensing layer over the electronic circuitlayer. The photo sensing layer includes a substrate layer and aphotodiode in contact with the electronic circuit layer. A lightreceiving surface is defined by a surface of the photodiode opposite tothe electronic circuit layer. The method can also include depositing acolor filter material over the photodiode. The method can also includeproviding a spot or well above the color filter material that is sizedand functionalized to receive a nucleic acid macromolecule, and toabsorb light from the nucleic acid macromolecule or to pass light to thelight receiving surface from the nucleic acid macromolecule.

A method of DNA sequencing according to some embodiments comprisesiteratively performing a process that may include labeling a nucleicacid macromolecule with a fluorescent label that identifies a nucleotidebase at a particular position in the nucleic acid macromolecule. Theprocess further includes detecting the fluorescent label associated withthe nucleic acid macromolecule. Detecting the fluorescent label includesilluminating the nucleic acid macromolecule with excitation light. Thenucleic acid macromolecule absorbs the excitation light and transmitsemitted light through a color filter and onto a photodiode of a backsideillumination complementary metal-oxide-semiconductor (CMOS) imagesensor. Detecting the fluorescent label further includes measuring atleast one parameter of the emitted light received at the photodiode.Detecting the fluorescent or chemiluminescent label further includescorrelating the at least one parameter of the emitted light to thefluorescent label. The process further includes removing the fluorescentlabel from the nucleic acid macromolecule. Without limitation, thebiosensors of embodiments of the invention may be used to carry outsequencing-by-synthesis (SBS), sequencing-by-ligation, cPAL sequencing,pyrosequencing, and combinations of the foregoing.

FIGS. 29-42 describe various stages of manufacture of a biosensoraccording to embodiments of the invention. Other embodiments ofmanufacture and configuration will be evident from this description tothose of skill in the art. It is therefore intended that the followingdescription be descriptive but not limiting.

For ease of reading, the text below is organized into sections. However,it will be understood that a description of subject matter in onesection (e.g., descriptions of macromolecules, filters, sequencingmethods, etc.) may also apply to subject matter in other sections.

Biosensors according to embodiments of the invention are not limited toa particular use. In one aspect, the biosensors of embodiments of theinvention find particular use for massively parallel DNA sequencing. DNAsequencing technologies are well known (see, e.g., Drmanac et al., 2010,“Human genome sequencing using unchained base reads on self-assemblingDNA nanoarrays,” Science 327:78-81; Shendure & Ji, (2008,“Next-generation DNA sequencing,” Nature Biotechnology 26:1135-45) andare therefore described only in general terms in sections below. Thefollowing paragraphs provide a brief initial discussion of sequencingand associated terminology so that certain features of the biosensorsdescribed below may be more easily understood.

A variety of DNA sequencing methods are known. In many approaches, largemolecules (e.g., genomic DNA) are broken into many smaller fragments,each having a characteristic DNA sequence. In array based technologies,the fragments are distributed to an array of positions on a substrate sothat each position in the array contains a DNA fragment with a singlecharacteristic sequence. Sequence information (“reads”) is obtained fromDNAs at each of thousands, or more often, millions, of positionssimultaneously and assembled by a computer. In most sequencingapproaches, the fragments are amplified prior to sequence determination.The amplification may occur prior to the positioning of the fragments ateach position, after the positioning of the fragments at each position,or both before and after positioning. The amplification step(s) produce“amplicons” which serve as “templates” in a sequencing process. Thus,for illustration, amplification may use RCA to produce a single-strandedconcatemer (e.g., a DNA nanoball) at each position on the array or usebridge PCR to produce a clonal population (or cluster) of DNA moleculeswith the same sequence at each position.

It will be understood that reference to a “DNA macromolecule,” and thelike, encompasses DNA nanoballs, branched structures, and clusteredclonal populations (i.e., more than a single molecule) or theirprecursors. In addition, a “DNA macromolecule,” and the like, mayencompass auxiliary DNA molecules such as primers and growing strandsproduced by primer extension or other processes encompasses. In manysequencing technologies, it is the auxiliary DNA molecules that comprise(or are “labeled” with) a detectable (e.g., fluorescent orchemiluminescent) dye that emit light detected by photodiodes of thebiosensor. Thus, a phrase such as “illuminating the nucleic acidmacromolecule with an excitation light source and detecting lightemitted from the macromolecule” will be understood to encompass“exposing a DNA nanoball or clonal cluster and associated labeledauxiliary molecules with an excitation light source and detecting lightemitted from the dyes of the labeled auxiliary molecules.”

In array-based sequencing methods, and the biosensors of embodiments ofthe invention, DNA macromolecules are positioned on a substrate in wellsor on “spots.” The wells or spots are able to receive and retain themacromolecule. Often, the spots, sometimes called “discrete spaced apartregions” or “pads”, comprise a substrate functionalized to receive anucleic acid macromolecule and the spots are separated by areas that are“inert” in the sense that DNA macromolecules do not bind such areas. Forexample, and without limitation, see Drmanac 2010, supra. “Wells” are atype of spot comprising walls that form a boundary or barrier to the DNAmacromolecules. Except where clear from context, reference to “spots”below may include wells.

In biosensors of embodiments of the invention, spots generally haveuniform dimensions and are organized as a regular (i.e., not random)array. The spots of an array are generally organized in a rectilinearpattern, often in columns and rows, but other regular patterns may beused (e.g., a spiral). The spots of an array may have characteristicdimensions, pitch, and density. The spots themselves may be circular,square, hexagonal or other shape. In the discussion below, the spots aregenerally assumed to be circular (i.e., can be described as having adiameter). It will be understood that reference to a “diameter” can alsorefer to linear dimensions of other shaped spots (e.g., diagonal, lengthor width). Thus, as used herein, “linear dimension” can refer to adiameter of a circle, width of a square, diagonal, and the like. In thecontext of biosensors of embodiments of the invention, the size of thespots is meaningful in two ways. First, the spots may be sized and/orfunctionalized in a way that limits occupancy to a single targetsequence. This may be a single DNA nanoball (a concatemer of a singletarget sequence) or a clonal cluster with a single target sequence. See,e.g., U.S. Pat. No. 8,133,719 and U.S. Pat. App. Pub. No. 2013/0116153,both incorporated by reference in their entireties for all purposes.Secondly, generally the spots may be sized and positioned relative tounderlying photodiodes so that each photodiode receives emitted lightfrom a single spot. In some embodiments, an array of spots may bepositioned over an array of corresponding photodiode(s) (and/or colorfilters) with a 1 to 1 correlation. That is, light emitted from an,e.g., DNA macromolecule at individual spot passes into an underlyingfilter and light not blocked by the filter is detected by a singlephotodiode associated with the filter, or light emitted from an, e.g.,DNA macromolecule, at individual spot passes into a plurality ofunderlying filters, each associated with a filter (specific forparticular wavelengths), each associated with a single photodiode, andlight not blocked by a filter is detected by the associated photodiode.Thus, as also discussed below, in some embodiments, light emitted from asingle spot may be detected by more than one photodiode (e.g., 2, 3, 4,etc.) photodiodes. In these embodiments, a group of multiple photodiodesassociated with a single spot may be referred to as a “unit cell” ofphotodiodes. The spots and filters (e.g., single filters or unit cells)may be arranged in the biosensor such that each photodiode in the unitcell receives light emitted from the same single spot. In addition, insome embodiments, the area of the light receiving surface of aphotodiode, or combined area of the light receiving surfaces of multiplephotodiodes associated with the same spot, is less than the area of thespot (from which light is emitted). Put another way, the spot may besmaller than the underlying photodiode(s) such that the boundary of thespot, if projected onto the light receiving surface of thephotodiode(s), is contained within the light receiving surface.

As is well known, nucleic acid sequencing generally involves aniterative process in which a fluorescent or chemiluminescent label isassociated in a sequence in a specific way with the DNA template(amplicon) being sequenced, the association is detected, and the labelis removed in the sense that it no longer emits a signal. See, e.g.,U.S. Pat. App. Pub. No. 2016/0237488; U.S. Pat. App. Pub. No.2012/0224050; U.S. Pat. Nos. 8,133,719; 7,910,354; 9,222,132; 6,210,891;6,828,100, 6,833,246; and 6,911,345, herein incorporated by reference intheir entireties. Thus it will be appreciated that, for example,“labeling a nucleic acid macromolecule with a fluorescent label” mayrefer to associating a labeled auxiliary molecule(s) with a DNA templateimmobilized on a spot.

Turning now to the drawings, FIG. 29 is a cross-sectional view of abackside illumination (BSI) CMOS image sensor 2900 according to someembodiments. The BSI CMOS image sensor 2900 may include a firstdielectric layer 2910. Although described as being dielectric, it iscontemplated that the first dielectric layer 2910 may include anysuitable electrically insulating material. The first dielectric layer2900 may include metal wiring 2913. The metal wiring 2913 may includeintegrated circuit materials and external connections. Together, thefirst dielectric layer 2900 and the metal wiring 2913 may becollectively referred to herein as an “electronic circuit layer” of theBSI CMOS image sensor.

A substrate layer 2915 may be provided over the first dielectric layer2910 and the metal wiring 2913. The substrate layer 2915 may be made ofany suitable material, such as, for example, silicon, III-V group onsilicon, graphene-on-silicon, silicon-on-insulator, combinationsthereof, and the like. The substrate layer 2915 may include openings inwhich light sensing components (e.g., photodiodes 2917) may bepositioned. Although described herein with respect to photodiodes 2917,it is contemplated that any suitable light sensing component may beused. The photodiodes 2917 may be configured to convert measured lightinto current. Photodiodes 2917 may include the source and drain of a MOStransistor (not shown) that may transfer the current to othercomponents, such as other MOS transistors. The other components mayinclude a reset transistor, a current source follower or a row selectorfor transforming the current into digital signals, and the like.Together, the substrate layer 2915 and the photodiodes 2917 may becollectively referred to herein as a “photo sensing layer” of the BSICMOS image sensor.

The photodiodes 2917 may be in contact with metal wiring 2913 tocommunicate the digital signals to external connections via the metalwiring 2913. In the BSI CMOS image sensor 2900 illustrated in FIG. 29,the light receiving surface is positioned at the top of the photodiodes2917 (i.e., on a surface not in contact with the electronic circuitlayer and opposite to the electronic circuit layer), and incident lightis received by the photodiodes 2917 at this light receiving surface.

According to FIG. 30, in order to construct a biosensor 3000, a firstpassivation layer 2920 may be deposited by conventional semiconductorprocessing techniques (e.g., low temperature plasma chemical vapordeposition) on the substrate layer 2915 and the photodiodes 2917 of theBSI CMOS image sensor 2900. The first passivation layer 2920 may includeany suitable protective material. For example, the first passivationlayer 2920 may include materials such as silicon, oxide, metals,combinations thereof, and the like. The first passivation layer 2920 mayact as an etch stop for later etching steps, as described furtherherein. The first passivation layer 2920 may alternatively oradditionally act to protect the active device (i.e., the backsideillumination CMOS sensor). The first passivation layer 2920 mayalternatively or additionally act to protect photodiodes 2917 from wearcaused by frequent use. The first passivation layer 2920 may betransparent. In one example, the first passivation layer 2920 may have athickness of 100 nanometers or less.

A. Biosensor 3000 of FIG. 30

FIG. 30 illustrates a biosensor 3000 that may be used for biological orchemical analysis (e.g., to detect the chemiluminescence of amacromolecule or macromolecular complex), according to some embodiments.The biosensor 3000 includes a backside illumination CMOS image sensor2900. The backside illumination CMOS image sensor 2900 includes anelectronic circuit layer (comprised of the first dielectric layer 2910and the metal wiring 2913) and a photo sensing layer over the electroniccircuit layer (comprised of a substrate layer 2915 and photodiodes2917). The photodiodes 2917 may be in contact with the electroniccircuit layer such that electronic signals may be transmitted from thephotodiode 2917 to the electronic circuit layer, and in someembodiments, to an external device. A light receiving surface is definedby a surface of the photodiodes 2917 that is opposite to the electroniccircuit layer (i.e., the surface in contact with the first passivationlayer 2920).

The biosensor 3000 may further include the first passivation layer 2920over the backside illumination CMOS image sensor 2900, and spots orwells (not shown) formed over or in the first passivation layer 2920 onor over which chemical or biological samples may be placed for analysis.In some embodiments, the biosensor 3000 may be adapted for detecting anoptical signal (e.g., fluorescent or chemiluminescent emission) from acorresponding array of biomolecules, where individual biomolecules maybe positioned over (e.g., in spots or wells) one or more photodiodessuch that the one or more photodiodes receive light from thebiomolecule, as discussed in greater detail below.

Various further embodiments for constructing biosensors using a backsideillumination CMOS sensor 3000 may now be described. According to FIG.31, a first metal layer 2923A may be deposited by conventionalsemiconductor processing techniques on the first passivation layer 2920of biosensor 3000 (e.g., by metal deposition techniques). The firstmetal layer 2923A may include any suitable metal material. For example,the first metal layer 2923A may include materials such as tungsten,aluminum, gold, copper, combinations or alloys thereof, and the like. Insome embodiments, the first metal layer 2923A may be a thick layer,e.g., thicker than the first passivation layer 2920. For example, thefirst metal layer 2923A may be up to 3 micrometers.

According to FIG. 32, the first metal layer 2923A may be etched toprovide first openings above the photodiodes 2917, leaving first metallayer 2923B. The first metal layer 2923A may be etched by any suitableprocess, such as, for example, wet etching, dry etching, combinationsthereof, and the like. It is contemplated that etching the first metallayer 2923A may involve use of a mask, for example. The etching may becompleted using any of a variety of materials, such as, for example,acids (e.g., hydrochloric acid, hydrofluoric acid, nitric acid, etc.),alkali with oxidizers, combinations thereof, and the like. It iscontemplated that the type of acid needed to etch the first metal layer2923A may depend on the material used for forming the first metal layer2923A. In some embodiments, the first openings may be aligned center tocenter with the photodiodes 2917, maximizing efficiency of thephotodiodes 2917 in later use. A mask (not shown) may define theopenings over the photodiodes 2917, leaving first metal layer 2923Bremaining, and the first passivation layer 2920 may act as an etch stopwhen etching the openings in the first metal layer 2923A. As describedherein, the pillars of the first metal layer 2923B may separate lightreceived by separate color filters and may reflect back light intendedfor a certain color filter back into that color filter or into thecorresponding photodiode 2917.

According to FIG. 33, a second dielectric layer 2925 may be depositedover the first metal layer 2923B and in the first openings byconventional semiconductor processing techniques. In some embodiments,the second dielectric layer 2925 may be formed on all exposed sides ofthe first metal layer 2923B. Although described as being dielectric, itis contemplated that the second dielectric layer 2925 may include anysuitable electrically insulating material, such as silicon nitride,tantalum oxide, combinations thereof, and the like. The seconddielectric layer 2925 may be formed of a same or different material thanthe first dielectric layer 2910.

According to FIG. 34, color filter material 2927A may be deposited overthe second dielectric layer 2925. In some embodiments, color filtermaterial 2927A may be deposited by spin coating. Color filter material2927A fills the openings created by second dielectric layer 2925. Inthis embodiment, color filter material 2927A is also deposited on theportions of second dielectric layer 2925 between the openings. Thus,according to FIG. 35, the excess color filter material 2927A above theopenings of the second dielectric layer 2925 may be removed, such as by,for example, chemical-mechanical planarization (CMP), leaving colorfilter material 2927B in the openings of the second dielectric layer2925.

However, it is also contemplated that in some embodiments, the colorfilter material may be formed using an alternative process. For example,as in FIG. 35, color filter material 2927B may be selectively depositedonly in the openings of the second dielectric layer 2925, such that morethan one (e.g., 2, 3 or 4) different color filter material 2927B may beplaced above the photodiodes 2917. In some applications, each differentcolor filter material 2927B may be associated with a separate photodiode2917.

The color filter material 2927B may include, for example, apigment-based polymer, a pigment-based dye, a dye-based polymer, a resinor other organic based material, combinations thereof, and the like.Color filter material 2927B may be necessary for the biosensor, forexample, because the photodiodes 2917 may alone detect light intensitywith little or no wavelength specificity, and thus cannot separate colorinformation.

Color filter material 2927B may include blue filter material, red filtermaterial, green filter material, emerald filter material, cyan filtermaterial, yellow filter material, magenta filter material, white filtermaterial, combinations thereof, and the like. Thus, the color filtermaterial 2927B may filter incident light by wavelength range, such thatthe separate filtered intensities include information about the color oflight. For example, red color filter material 2927B may give informationabout the intensity of light in red wavelength regions. Blue colorfilter material 2927B may give information about the intensity of lightin blue wavelength regions. Green color filter material 2927B may giveinformation about the intensity of light in green wavelength regions,and so on and so forth.

In some embodiments, color filter material 2927B may include material ofa single color. For example, each of the color filter materials 2927Bmay be red. In some embodiments, color filter material 2927B may includematerial of different colors, with each color filter material 2927Bcorresponding to a separate photodiode 2917. For example, one colorfilter material 2927B may be red, and a neighboring color filtermaterial 2927B may be green. FIG. 42A illustrates such an embodiment, inwhich a two-channel color filter is used. In FIG. 42A, a biological orchemical sample (e.g., a DNA macromolecule) may be positioned in a spotor well 1450 such that emissions from the macromolecule enter both thered color filter material 4227B and the green color filter material4227A (e.g., overlapping both the red color filter material 4227B andthe green color filter material 4227A), and such that the emittedwavelength of light through the different colors of the color filtermaterial may be detected. In another example, more than two surroundingcolor filter materials 127B may include material of different colors.FIG. 42B illustrates such an embodiment, in which a four-channel colorfilter is used. The four-channel color filter may include one colorfilter material 4227B that is red, one color filter material 4227D thatis yellow, one color filter material 4227A that is green, and one colorfilter material 4227C that is blue. In this example, a biological orchemical sample may be placed in a spot or well 4250 at the intersectionof the four color filters, such that the emitted wavelength of lightthrough the four colors of the color filter material may be detected. Insome embodiments, spot or well 4250 may lie above each of the underlyingcolor filter materials (and corresponding photodiodes) equally, i.e., sothat equal areas of each filter underlies the spot.

FIG. 36A illustrates an embodiment in which biosensor 3600 isconstructed. According to FIG. 36A, the second passivation layer 2930may be deposited according to conventional semiconductor techniques overthe second dielectric layer 2925 and the color filter material 2927B.The second passivation layer 2930 may be as described below with respectto FIG. 36B. A first material layer 2935 may be deposited over thesecond passivation layer 2930. The first material layer 2935 may includeany suitable materials, such as silicon nitride, tantalum oxide,combinations thereof, and the like. A second material layer 2937 may bedeposited over the first material layer 2935. The second material layer2937 may include any suitable materials, such as silicon dioxide and thelike. In some embodiments, the first material layer 2935 may have arefractive index that is higher than the refractive index of the secondmaterial layer 2937. In some embodiments, the first material layer 2935may have a refractive index that is higher than the second passivationlayer 2930. Thus, the embodiment of FIG. 36A may result in efficientdelivery of excitation light to the light receiving surface in the caseof fluorescence measurement. For example, the first material layer 2935may form the core of an optical waveguide, thus permitting low losstransmission of excitation light. In some embodiments, biological orchemical samples may be placed on the second material layer 2937 abovethe photodiodes 2917 (in some embodiments, in openings or wells formedon the second material layer 2937), and their fluorescence orchemiluminescence may be measured by the photodiodes 2917, as describedfurther herein. When measuring fluorescence in the embodiment shown inFIG. 36A, however, the excitation light may be directed sideways, alongthe surface of the biosensor 3600, in some examples.

B. Biosensor 3600 of FIG. 36A

Thus, FIG. 36A illustrates a biosensor 3600 that may be used forbiological or chemical analysis according to some embodiments. Thebiosensor 3600 may include a backside illumination CMOS image sensor2900. The backside illumination CMOS image sensor 2900 includes anelectronic circuit layer (comprised of the first dielectric layer 2910and the metal wiring 2913) and a photo sensing layer over the electroniccircuit layer (comprised of a substrate layer 2915 and photodiodes2917). The photodiodes 2917 may be in contact with the electroniccircuit layer such that electronic signals may be transmitted from thephotodiode 2917 to the electronic circuit layer, and in someembodiments, to an external device. A light receiving surface is definedby a surface of the photodiodes 2917 that is opposite to the electroniccircuit layer (i.e., the surface in contact with the first passivationlayer 2920).

The biosensor 3600 may further include the first passivation layer 2920over the backside illumination CMOS image sensor 2900, and a first metallayer 2923B over the first passivation layer 2920. The first metal layer2923B may also be positioned over substrate layer 2915. The first metallayer 2923B may include first openings. The biosensor 3600 may furtherinclude a second dielectric layer 2925 over the metal layer 2923B andthe first passivation layer 2920. The second dielectric layer 2925 mayalso be positioned in the first openings of metal layer 2923B.

The biosensor 3600 may further include color filter material 2927B overthe second dielectric layer 2925 and in and above the first openings ofmetal layer 2923B, such that a top surface of color filter material2927B may be planar with a top surface of the second dielectric layer2925 over the metal layer 2923B. The biosensor 3600 may further includea second passivation layer 2930 over the second dielectric layer 2925and the color filter material 2927. The biosensor 3600 may furtherinclude a first material layer 2935 and a second material layer 2937.The first material layer 2935 may have a higher refractive index thanthe second material layer 2937. A biological or chemical sample may beplaced in spots or wells (not shown) formed in or on the second materiallayer 2937 for analysis, as described further herein.

FIG. 36B illustrates an alternative embodiment than FIG. 36A. Accordingto FIG. 36B, a second passivation layer 2930 may be deposited accordingto conventional semiconductor techniques over the second dielectriclayer 2925 and the color filter material 2927B. The second passivationlayer 2930 may include any suitable materials, such as, for example,silicon nitride, tantalum oxide, combinations thereof, and the like. Insome embodiments, the second passivation layer 2930 may include one ormore high-k materials. The second passivation layer 2930 may include thesame or different materials than the first passivation layer 2920. Insome embodiments, the second passivation layer 2930 is made of a densermaterial than the first passivation layer 2920. The second passivationlayer 2930 may, in some embodiments, act as a protective materialbetween a sample being analyzed and the color filter material 2927B. Insome embodiments, the second passivation layer 2930 acts as an etch stopfor later etching steps. The second passivation layer 2930 may betransparent.

Further according to FIG. 36B, a second metal layer 2933A may bedeposited according to conventional semiconductor techniques over thesecond passivation layer 2930. The second metal layer 2933A may includeany suitable metal material, such as, for example, tungsten, aluminum,copper, combinations thereof, and the like. The second metal layer 2933Amay be made of the same or a different material than the first metallayer 2923B. The second metal layer 2933A may be opaque to incident orexcitation light.

Then, according to FIG. 37, the second metal layer 2933B may be etchedout of the second metal layer 2933A or patterned, creating secondopenings 2950A-C in the second metal layer 2933A. In some embodiments,the second openings 2950A-C may be aligned center to center with thephotodiodes 2917. In some embodiments, the second openings 2950A-C mayhave a diameter in the range of 100 nanometer to 1 micrometer. Thesecond openings 2950A-C may have a smaller width or diameter than thecolor filter material 2927B. In some embodiments, biological or chemicalsamples may be placed in the second openings 2950A-C, and light emittedfrom the samples may be used to measure their fluorescence orchemiluminescence, as described further herein. In embodiments in whichthe second openings 2950A-C are smaller in width or diameter than thecolor filter material 2927B, there may be increased blockage of incidentor excitation light, resulting in less noise in detection of thefluorescence or luminescence of a sample. The width or diameter of thesecond openings 2950A-C may approximately correspond to the size of thebiological or chemical sample being analyzed.

C. Biosensor 3700 of FIG. 37

Thus, FIG. 37 illustrates a biosensor 3700 that may be used forbiological or chemical analysis according to some embodiments. Thebiosensor 3700 includes a backside illumination CMOS image sensor 2900.The backside illumination CMOS image sensor 2900 includes an electroniccircuit layer (comprised of the first dielectric layer 2910 and themetal wiring 2913) and a photo sensing layer over the electronic circuitlayer (comprised of a substrate layer 2915 and photodiodes 2917). Thephotodiodes 2917 may be in contact with the electronic circuit layersuch that electronic signals may be transmitted from the photodiode 2917to the electronic circuit layer, and in some embodiments, to an externaldevice. A light receiving surface is defined by a surface of thephotodiodes 2917 that is opposite to the electronic circuit layer (i.e.,the surface in contact with the first passivation layer 2920).

The biosensor 3700 may further include the first passivation layer 2920over the backside illumination CMOS image sensor 2900, and a first metallayer 2923B over the first passivation layer 2920. The first metal layer2923B may also be positioned over substrate layer 2915. The first metallayer 2923B may include first openings. The biosensor 3700 may furtherinclude a second dielectric layer 2925 over the metal layer 2923B andthe first passivation layer 2920. The second dielectric layer 2925 mayalso be positioned in the first openings of metal layer 2923B.

The biosensor 3700 may further include color filter material 2927B overthe second dielectric layer 2925 and in and above the first openings ofmetal layer 2923B, such that a top surface of color filter material2927B may be planar with a top surface of the second dielectric layer2925 over the metal layer 2923B. The biosensor 3700 may further includea second passivation layer 2930 over the second dielectric layer 2925and the color filter material 2927. The biosensor 3700 may furtherinclude a second metal layer 2933B having second openings 2950A-C. Thesecond openings 2950A-C may function as spots or wells configured toreceive biological or chemical samples, as described further herein.

Referring again to the embodiment of FIG. 37, various furthermanufacturing techniques may be implemented for further signalenhancement, as described herein with respect to FIGS. 38-41. Accordingto FIG. 38, microlenses 2940A may be grown over the second passivationlayer 2930 and the second metal layer 2933B. In some embodiments, themicrolenses 2940A may be aligned center to center with the photodiodes2917. The microlenses 2940A may include a variety of materials, such asglass, polymers, plastics, combinations thereof, and the like. Themicrolenses 2940A may be included in the device above each of the colorfilters 2927B to focus light emitted into each of the color filters2927B.

The microlenses 2940A may be grown according to any suitable microlensfabrication process, such as those commonly used with respect to CMOSimage sensors. As one example, photolithography may be performed on aphotoresist or ultraviolet curable epoxy material and the material maybe melted to form arrays of microlenses 2940A. As another example, smallfilaments of glass may be melted, and the surface tension of the moltenglass may form smooth spherical surfaces. The spherically-surfaced glassmay then be mounted and grinded as appropriate to form microlenses2940A. In still another example, wafer-level optics (WLO) may be used,in which multiple lens wafers are precision aligned, bonded together,and diced to form multi-element stacks that may be used as microlenses2940A.

According to FIG. 39, a third metal layer 2943A may be depositedaccording to conventional semiconductor processing techniques over themicrolenses 2940A. The third metal layer 2943A may include any suitablematerials, such as tungsten, aluminum, copper, combinations thereof, andthe like. The third metal layer 2943A may be a relatively thin layer,e.g., thinner than the second metal layer 2923B. The third metal layer2943A may be made of the same or different materials than the firstmetal layer 2923B and/or the second metal layer 2933B.

According to FIG. 40, a planarization layer 2945A may be deposited overthe third metal layer 2943A. The planarization layer 2945A may includeany suitable materials. The planarization layer 2945A may be depositedby, for example, spin coating, or by any other suitable method. If theplanarization layer 2945A exceeds a top exposed surface of the thirdmetal layer 2943A, the planarization layer 2945A may be planarized by,for example, chemical-mechanical planarization (CMP), leaving theplanarization layer 2945A in the openings between the third metal layer2943A and creating a substantially planar upper surface.

According to FIG. 41, third openings 2955A-C may be etched throughplanarization layer 2945A (leaving planarization layer 2945B remaining),the third metal layer 2943A (leaving the third metal layer 2943Bremaining), and microlenses 2940A (leaving the microlenses 2940Bremaining). For example, the planarization layer 2945B may be spincoated with a photoresist (not shown) in order to etch the thirdopenings 2955A-C. In some embodiments, the width of the third openings2955A-C may correspond to the width of the second openings 2950A-C, suchthat the second metal layer 2933B does not need to be further etched.Third openings 2955A-C may be etched to the second passivation layer2930, with the second passivation layer 2930 acting as an etch stop. Insome examples, third openings 2955A-C may have a diameter between 100nanometers and 1 micrometer, and may be aligned center to center withthe color filter material 2927B and/or the photodiode 2917. In someembodiments, biological or chemical samples may be placed in the thirdopenings 2955A-C on the second passivation layer 2930, and thefluorescence or chemiluminescence of the samples may be measured, asdescribed further herein.

D. Biosensor 4100 of FIG. 41

Thus, FIG. 41 illustrates a biosensor 4100 that may be used forbiological or chemical analysis according to some embodiments. Thebiosensor 4100 includes a backside illumination CMOS image sensor 2900.The backside illumination CMOS image sensor 2900 includes an electroniccircuit layer (comprised of the first dielectric layer 2910 and themetal wiring 2913) and a photo sensing layer over the electronic circuitlayer (comprised of a substrate layer 2915 and photodiodes 2917). Thephotodiodes 2917 may be in contact with the electronic circuit layersuch that electronic signals may be transmitted from the photodiode 2917to the electronic circuit layer, and in some embodiments, to an externaldevice. A light receiving surface is defined by a surface of thephotodiodes 2917 that is opposite to the electronic circuit layer (i.e.,the surface in contact with the first passivation layer 2920).

The biosensor 4100 may further include the first passivation layer 2920over the backside illumination CMOS image sensor 2900, and a first metallayer 2923B over the first passivation layer 2920. The first metal layer2923B may also be positioned over substrate layer 2915. The first metallayer 2923B may include first openings. The biosensor 4100 may furtherinclude a second dielectric layer 2925 over the metal layer 2923B andthe first passivation layer 2920. The second dielectric layer 2925 mayalso be positioned in the first openings of metal layer 2923B.

The biosensor 4100 may further include color filter material 2927B overthe second dielectric layer 2925 and in and above the first openings ofmetal layer 2923B, such that a top surface of color filter material2927B may be planar with a top surface of the second dielectric layer2925 over the metal layer 2923B. The biosensor 4100 may further includea second passivation layer 2930 over the second dielectric layer 2925and the color filter material 2927. The biosensor 4100 may furtherinclude a second metal layer 2933B over the second passivation layer2930 having second openings 2950A-C.

The biosensor 4100 may further include microlenses 2940B over the secondmetal layer 2933B, a third metal layer 2943B over the microlenses 2940B,and a planarization layer 2945 over the third metal layer 2943B. Thethird metal layer 2943B may serve a number of different purposes in thebiosensor 4100. For example, the third metal layer 2943B may help toblock incident light from entering the color filter material 2927B. Inaddition, because the third metal layer 2943B is curved, any lightemitted from a biological or chemical sample may be passed through themicrolenses 2940B, reflected off of the third metal layer 2943B, anddirected back toward the color filter material 2927B and thus, the lightreceiving surface of the photodiode 2917. In other words, the amount ofemitted light that may be measured by the photodiode 2917 may bemaximized.

The planarization layer 2945 may form a planar surface over the thirdmetal layer 2943B. The microlenses 2940B, the third metal layer 2943B,and the planarization layer 2945 may have third openings 2955A-C formedtherein that may overlap with the second openings 2950A-C in someembodiments. For example, the third openings 2955A-C may have the samewidth as the second openings 2950A-C. However, it is contemplated thatin some embodiments, the third openings 2955A-C may have a differentwidth than the second openings 2950A-C. Together, the second openings2950A-C and the third openings 2955A-C may function as spots or wellsconfigured to receive biological or chemical samples, as describedfurther herein. Because the third openings 2955A-C of FIG. 41 are deeperthan the second openings 2950A-C of FIG. 37, excitation light maygenerally be directed from a source positioned directly above the thirdopenings 2955A-C in biosensor 4100. Biosensor 3700 may be able totolerate more angular misalignment of excitation light because secondopenings 2950A-C are not as deep as third openings 2955A-C.

Nucleic Acid Sequencing Applications

As described above with respect to FIGS. 30, 36A, 37, and 41, biologicalor chemical samples may be placed on each of the described biosensorsabove color filter material 2927B and the photodiodes 2917. Thebiological or chemical sample may include any of a number of components.For example, the sample may contain nucleic acid macromolecules (e.g.,DNA, RNA, etc.), proteins, and the like. The sample may be analyzed todetermine a gene sequence, DNA-DNA hybridization, single nucleotidepolymorphisms, protein interactions, peptide interactions,antigen-antibody interactions, glucose monitoring, cholesterolmonitoring, and the like.

As discussed above, in some embodiments the biomolecule is a nucleicacid, such as DNA. Without limitation, the DNA biomolecule may be a DNAnanoball (single stranded concatemer) hybridized to labeled probes(e.g., in DNB sequencing by ligation or cPAL methods) or tocomplementary growing strands (e.g., in DNB sequencing by synthesismethods) or both; or to a single DNA molecule (e.g., in single moleculesequencing); or to a clonal population of DNA molecules, such as iscreated in bridge PCR based sequencing. Thus, reference to “abiomolecule”, “a DNA macromolecule” or “a nucleic acid macromolecule”may encompass more than one molecule (e.g., a DNB associated withmultiple growing complementary strands or a DNA cluster comprisingclonal population of hundreds or thousands of DNA molecules). See, e.g.,U.S. Pat. No. 8,133,719; U.S. Pat. App. Pub. No. 2013/0116153, U.S. Pat.App. Pub. No. 2016/0237488; U.S. Pat. App. Pub. No. 2012/0224050; U.S.Pat. Nos. 8,133,719; 7,910,354; 9,222,132; 6,210,891; 6,828,100,6,833,246; and 6,911,345, herein incorporated by reference in theirentireties.

In some embodiments, color filter material 2927B may be sized andfunctionalized to receive (in spots or wells above color filter material2927B) biological or chemical samples and to absorb light emitted fromthe biological or chemical sample in some examples. For example, ifcolor filter material 2927B is red and the emitted light from thebiological or chemical sample is green, color filter material 2927B mayabsorb the green emitted light. In some embodiments, color filtermaterial 2927B may be sized and functionalized to receive (in spots orwells above color filter material 2927B) biological or chemical samplesand to pass light emitted from the biological or chemical sample throughthe color filter material 2927B and onto the light receiving surface ofthe photodiode 2917. For example, if color filter material 2927B is blueand the emitted light from the biological or chemical sample is blue,color filter material 2927B may pass the blue emitted light through tothe light receiving surface of the corresponding photodiode 2917. Inother words, in some embodiments, emitted light may be absorbed by colorfilter material 2927B. In some embodiments, emitted light may betransmitted through the color filter material 2927B and onto thephotodiode 2917.

To achieve high density and assist in alignment between the nucleic acidmacromolecules and the photodiodes 2917 of the biosensor, the surface ofthe biosensor may be constructed such that there are active spots orwells (e.g., openings 2950A-C, openings 2955A-C, etc.) that are sizedand chemically functionalized to receive a nucleic acid macromolecule,surrounded by areas of the surface to which the nucleic acidmacromolecules may not bind. The nucleic acid macromolecules may besecured to the active surface aligned with the photodiode 2917 using anysuitable surface chemistry. This may include non-covalent interaction(for example, to an area bearing positive charge) or interaction with acapture probe or oligonucleotide attached to the surface, bearing asequence that is complementary to a sequence contained in the nucleicacid macromolecule. See, for example, U.S. Pat. No. 8,445,194, which isherein incorporated by reference in its entirety.

Example

This example demonstrates that BSI CIS sensors may be used to detectweak signals from surface attached photon emitting molecules. Weconstructed a biosensor as described in FIG. 37, but without a colorfilter layer (i.e., lacking elements 2920, 2923B, 2925, and 2927B). Inaddition, surface 2933B was rendered hydrophobic, and the bottomsurfaces of openings 2950A/B/C were rendered hydrophilic (such that DNBswere distributed toward the hydrophilic surfaces and away from thehydrophobic surfaces).

A dilute solution of DNA nanoballs (DNBs) was applied the biosensorarray allowing individual DNBs to settle on the spots of the array. Forpurposes of this experiment all of the DNBs have the same sequence, incontrast to sequencing methods in which essentially all DNBs on an arraywill have different sequences, and in which the sequence of a DNB areany specific spot/position will not be known prior to sequencedetermination.

Two primers were hybridized to the DNA templates (see FIG. 43A, top).The “left” primer has a blocked (nonextendible) 3′ terminus and islabeled at the 5′ terminus with a fluorescent dye. The fluorescent dyewas used to establish the position of the DNBs on the array (not shown).The “right” primer acts as an extendible primer for sequencing bysynthesis. Sequencing reagents and detection reagents 4 were added (DNApolymerase, streptavidin, biotinylated luciferase 3, ATP and luciferin)along with dATP tagged with biotin via a cleavable linker. In thissystem the streptavidin 2 associates with the biotin conjugated to theincorporated nucleotide and also associates with biotinylatedluciferase, as shown in FIG. 43A. (Biotin 1 symbolized by diamond.) TheATP acts as a substrate for the generation of light by theluciferase-mediated conversion of luciferin to oxyluciferin. The lightis received by the photodiodes, generating a signal. The signal iscorrelated with incorporation of dATP, indicating the present of thymineat the corresponding position of the template sequence. FIG. 43A showssignal from DNBs at numerous spots on the array.

THPP was then used to cleave the cleavable linker, releasing thebiotin/streptavidin/luciferase complex, and the array was washed toremove all soluble reagents. FIG. 43B shows that following the wash stepsignal from the array is absent or significantly reduced.

A second incorporation round was carried out using dTTP-digoxin and DNApolymerase as shown in FIG. 43C. The incorporation of dTTP is detectedusing a biotinylated anti-digoxin antibody, streptavidin, biotinylatedluciferase, ATP and luciferin. The use of biotinylated anti-digoxinantibody amplifies the signal generated by each incorporation event.FIG. 43C is an image showing that chemiluminscent light was generated atnumerous spots on the array. This example demonstrates, using twodifferent dNTPs and two different detection systems, that the BSI CISsensors of the invention may be used to detect weak signals from surfaceattached photon emitting molecules such as DNBs.

VII. Alternative Differential Surfaces for a Biosensor

FIGS. 44-47 describe various stages of manufacture of a biosensor havingdifferential surfaces according to embodiments of the invention. Otherembodiments of manufacture and configuration will be evident from thisdescription to those of skill in the art. It is therefore intended thatthe following description be descriptive but not limiting.

FIG. 44 is a cross-sectional view of a backside illumination (BSI) CMOSimage sensor with the mask removed, according to some embodiments.According to FIG. 44, voids 4460A-C may be created by the metal layer ormetal oxide layer 4433B on the sides, and the passivation layer 4420 onthe bottom. The device structure in FIG. 44 is similar to device 2600 inFIG. 26, but with a substrate including sensors 4417 and a metal wiring4413 in a dielectric layer 4410. The device structure in FIG. 44 is alsosimilar to top portion of device 200 in FIG. 2. The device structure inFIG. 44 is also similar to top portion of device 3700 in FIG. 37, withthe filter layer removed. The method described here is also applicableto similar device structures. The voids 4460A-C may form a spot or wellinto which biological or chemical samples may be placed, as describedfurther herein.

In some embodiments, a first covering layer and a second covering layer,different from the first covering layer, may be selectively appliedbased on the differential surfaces of the metal layer or metal oxidelayer 4433B and the passivation layer 4420, respectively. The first andsecond covering layers have different properties, resulting in an arrayof spots or wells comprising a bottom surface comprising the secondcovering layer, separated by areas (e.g., 4433B) comprising the firstcovering layer. In some embodiments macromolecules of interestpreferentially associate with the second covering layer compared withthe first covering layer.

FIG. 45 is a cross-sectional view of a backside illumination CMOS imagesensor with a first coating selectively applied due to differentialsurfaces, according to some embodiments. As illustrated in FIG. 45, thefirst covering layer 4450 may be selectively applied to the metal layeror metal oxide layer 4433B based on its surface properties. For example,the first covering layer 4450 may be of such a material that it may bondto and/or be attracted to the metal layer or metal oxide layer 4433B. Insome embodiments the first covering layer does not bind or adhere to, oris repelled by, the passivation layer 4420, resulting in the structureshown in FIG. 45. The first covering layer 4450 may be applied to themetal layer or metal oxide layer 4433B according to any method ortechnique (e.g., chemical vapor deposition, dipping, spin coating,and/or the like). For example, the metal layer or metal oxide layer4433B may be coated or treated with a first material to form the firstcovering layer 4450. The first covering layer 4450 may be depositedaccording to conventional semiconductor processing techniques. It willbe recognized that term “covering layer” is not intended to ascribe anyparticular structure or dimensions.

The first covering layer 4450 may include any suitable material thatadheres or binds the metal or metal oxide material 4433B. In oneapproach the first covering layer 4455 is produced by application of aphosphate compound that binds metal or metal oxide, including withoutlimitation, inorganic phosphate, phosphonic acid, organic phosphatecompounds such as hexamthethylphosphoramide, hexmamethyl tetraphospate,combinations thereof, and the like.

In some embodiments, the first covering layer 4450 may include amaterial that repels biological or chemical analytes of interest. Forexample, the first covering layer 4450 may include a material that has anegative charge, thus repelling negatively charged biological orchemical samples. In some embodiments, the first covering layer 4450 maybe hydrophobic. Those of ordinary skill in the art will recognize thatcombinations (e.g., pairwise combinations) of metals and the firstcovering layer can be selected and optimized for particular purposes.

FIG. 46 is a cross-sectional view of a backside illumination CMOS imagesensor with a second coating selectively applied due to differentialsurfaces, according to some embodiments. As illustrated in FIG. 46, thesecond covering layer 4455 may be selectively applied to the passivationlayer 4420 based on the surface properties of the passivation layer. Forexample, the second covering layer 4455 may be of such a material thatit may bond to and/or be attracted to the passivation layer 4420, butdoes not bond to or adhere to the first covering layer 4450 which coversmetal or metal oxide 4433B. The second covering layer 4455 may beapplied by coating or treating the exposed portions of the passivationlayer 4420 with a second material. In one approach, both the exposedpassivation layer 4420 and metal or metal oxide 4433B regions covered bythe first covering layer 4450 are exposed to the second material, whichadheres only on the passivation layer. The second covering layer 4455may be deposited according to conventional semiconductor processingtechniques.

In one approach the second covering layer 4455 is produced byapplication of silane or a silane compound, including withoutlimitation, aminopropyltrimethoxysilane,3-aminopropyl-methyldiethoxysilane, 3-aminopropyltri-ethoxysilane, etc.In some embodiments, the second covering layer 4455 may include amaterial that attracts biological or chemical samples. For example, thesecond covering layer 4455 may include a material that has a positivecharge, thus attracting negatively charged biological or chemicalsamples. In some embodiments, the second covering layer 4455 may behydrophilic. Those of ordinary skill in the art will recognize thatcombinations (e.g., pairwise combinations) of the first covering layerand the passivation layer 4420 (i.e., the surface of the passivationlayer) can be selected and optimized for particular purposes.

It will be recognized that the term “covering layer” is not intended tolimit the first and second covering layers to any particular method ofapplication or structure. As noted, different properties of the firstand the second covering layers may be selected to differentially retaintarget macromolecule(s), e.g., DNA macromolecules. It will also berecognized that the first and/or second covering layers may befunctionalized such that the functionalized surface has a property thatresults in differential retention of target macromolecule(s). Forillustration, after application of the first and second covering layersa DNA binding molecule (e.g., oligonucleotide) with affinity to thesecond covering layers, but not to the first covering layers, may beapplied to cover second covering layer 4455. In some embodiments, thesecond covering layer 4455 is a functionalized surface on which a singlenucleic acid molecule is amplified.

It will be recognized that the term “first covering layer” may refer tothe material applied to the surface as well as the material retained onthe surface (e.g., the latter may differ from the former by evaporationof a solvent; by a reaction with the surface material, and the like).

Thus, a structure may be created in which a first covering layer 4450Bis present on the metal layer or metal oxide layer 4433B, and a secondcovering layer 4455 is present in the voids 4460A-C. The voids 4460A-Cmay be formed by the first covering layer 4450B and the metal layer ormetal oxide layer 4433B on the sides, and the passivation layer 4420 onthe bottom. The voids 4460A-C may form a spot or well into whichbiological or chemical samples may be placed, as described furtherherein.

FIG. 47 is a cross-sectional view of a biosensor using a backsideillumination CMOS image sensor with macromolecules, according to someembodiments. According to FIG. 47, biological or chemical samples 4470may be introduced in the voids atop the second covering layer 4455. Theinvention is not limited to any particular method of introduction. Insome embodiments, the biological or chemical samples 4470 may beattracted to or bind to the second covering layer 4455, while beingrepelled by the first covering layer 4450. This may prevent thebiological or chemical samples 4470 from sticking to the first coveringlayer 4450B on the metal layer or metal oxide layer 4433B where theycannot be sensed by the photodiodes 4417.

As illustrated in FIGS. 44, 46, and 47, in some embodiments, the metallayer or metal oxide layer 4433B and the top surfaces of photodiodes4417 or the passivation layer 4420 form a plurality of wells or voids4460A-C, wherein wall(s) of each well are formed from the metal layer,and the bottoms of each well are formed from the photodiode 4417 surfaceor from the overlying passivation layer 4420. In some embodiments, thewall(s) may have a height, h, that extends from the well bottom to thelevel corresponding the top of covering layer 4450B where the bottom andwalls define the void(s) 4460A-C. In some embodiments, the surface areaof the well bottom is less that the surface area of the underlyingphotodiode. In some embodiments, the volume of the void 4460A-C is inthe range of 1×10-24 m3-1×10-21 m2; and/or the height of the walls is inthe range of 1 nm-500 nm; and/or the area of the bottom is in the rangeof 1×10-15 m2-1×10-14 m2. In some embodiments, the ratio of the width ordiameter of the well to the height of the walls is in the range of1-200.

In some embodiments, as illustrated in FIG. 47, a biosensor 4700 caninclude a backside illumination complementary metal-oxide-semiconductor(CMOS) image sensor 4411. Backside illumination CMOS image sensor 4411can include an electronic circuit layer 4412 and a photo sensing layer4414 over the electronic circuit layer. Electronic circuit layer 4412can be comprised of a dielectric layer 4410 and metal wiring 4413. Photosensing layer 4414 can include a substrate layer 4415 and a plurality ofphotodiodes 4417 having a first top surface 4417A and a first bottomsurface 4417B. The first bottom surface 4417B can be in contact with theelectronic circuit layer 4413 (connections not explicitly shown), andthe first top surface 4417A includes a light receiving surface.Biosensor 4700 can also have a metal or metal oxide layer 4433B over thephoto sensing layer 4414, and the metal or metal oxide layer 4433B has asecond top surface 4433-1 and a second bottom surface 4433-2. The metalor metal oxide layer 4433B defines a plurality of voids 4460, and eachvoid of the plurality of voids 4460 can be aligned with at least onephotodiode of the plurality of photodiodes 4417. The second top surface4433-1 can be coated or treated with a first material 4450 to form afirst covering layer. Biosensor 4700 can also have a passivation layer4420 over the plurality of photodiodes 4417, and the passivation layerhas a third top surface 4420A and a third bottom surface 4420B. Themetal or metal oxide layer 4433B and the third top surface 4420A of thepassivation layer 4420 form a plurality of wells 4465. The walls of eachwell are formed from the metal or metal oxide layer 4433B, and thebottom of each well is formed from the third top surface 4420A of thepassivation layer 4420. The bottom of each well can be coated or treatedwith a second material 4455 to form a second covering layer. The firstmaterial 4450 is different than the second material 4455.

In some embodiments of biosensor 4700, the first material can include atleast one of phosphate or phosphonic acid. The second material caninclude silane. In some embodiments, the plurality of wells arefunctionalized to receive macromolecules. In some embodiments, themacromolecules are less likely to bind to the first material than to thesecond material. In some embodiments, the second material is configuredto bind the macromolecules, and the first material is configured not tobind to the macromolecules. In some embodiments, the second material caninclude a ligand that binds the macromolecules. Without limitation, themacromolecule can be a nucleic acid, protein (e.g., antigen), orantibody, and the ligand can be an oligonucleotide, DNA binding protein,antigen, or antibody. The macromolecules can be antibodies that bind aDNA macromolecule. In some embodiments, the first material ishydrophobic, and the second material is hydrophilic. At least one wellcan be occupied by a macromolecule analyte. The macromolecule analytecan be a nucleic acid or antibody.

The biological or chemical samples may include any of a number ofcomponents. For example, a sample may contain nucleic acidmacromolecules (e.g., DNA, RNA, etc.), proteins, and the like. Thesample may be analyzed to determine a gene sequence, DNA-DNAhybridization, single nucleotide polymorphisms, protein interactions,peptide interactions, antigen-antibody interactions, glucose monitoring,cholesterol monitoring, and the like.

Although the processes described herein are described with respect to acertain number of steps being performed in a certain order, it iscontemplated that additional steps may be included that are notexplicitly shown and/or described. Further, it is contemplated thatfewer steps than those shown and described may be included withoutdeparting from the scope of the described embodiments (i.e., one or someof the described steps may be optional). In addition, it is contemplatedthat the steps described herein may be performed in a different orderthan that described.

In the foregoing description, aspects of the application are describedwith reference to specific embodiments thereof, but those skilled in theart will recognize that the invention is not limited thereto. Thus,while illustrative embodiments of the application have been described indetail herein, it is to be understood that the inventive concepts may beotherwise variously embodied and employed, and that the appended claimsare intended to be construed to include such variations, except aslimited by the prior art. Various features and aspects of theabove-described invention may be used individually or jointly. Further,embodiments can be utilized in any number of environments andapplications beyond those described herein without departing from thebroader spirit and scope of the specification. The specification anddrawings are, accordingly, to be regarded as illustrative rather thanrestrictive. For the purposes of illustration, methods were described ina particular order. It should be appreciated that in alternateembodiments, the methods may be performed in a different order than thatdescribed.

Other variations are within the spirit of the present disclosure. Thus,while the disclosed techniques are susceptible to various modificationsand alternative constructions, certain illustrated embodiments thereofare shown in the drawings and have been described above in detail. Itshould be understood, however, that there is no intention to limit thedisclosure to the specific form or forms disclosed, but on the contrary,the intention is to cover all modifications, alternative constructionsand equivalents falling within the spirit and scope of the disclosure,as defined in the appended claims.

1. A method for forming sequencing flow cells, comprising: providing asemiconductor wafer covered with a dielectric layer; forming a patternedlayer on the dielectric layer, the patterned layer having differentialsurface regions that include first surface regions and second surfaceregions; attaching a cover wafer to the semiconductor wafer to form acomposite wafer structure that includes a plurality of sequencing flowcells, wherein each sequencing flow cell includes: a flow channelbetween the patterned layer and the cover wafer; one or more firstsurface regions in the patterned layer; one or more second surfaceregions in the patterned layer; and an inlet and an outlet coupled tothe flow channel; and singulating the composite wafer structure to forma plurality of dies, each die including a sequencing flow cell.
 2. Themethod of claim 1, wherein the first surface regions are hydrophilicsurfaces and the second surface regions are hydrophobic surfaces.
 3. Themethod of claim 1, wherein the first surface regions hydrophobic aresurfaces and the second surface regions are hydrophilic surfaces.
 4. Themethod of claim 1, further comprising forming a plurality of throughholes in the semiconductor wafer before attaching the cover wafer, theplurality of through holes configured as inlets and outlets for the flowcells.
 5. The method of claim 1, further comprising forming inlets andoutlets in the cover wafer before attaching the cover wafer to thesemiconductor wafer.
 6. The method of claim 1, wherein the semiconductorwafer further comprises a CMOS layer underlying the dielectric layer. 7.The method of any of claim 1, wherein forming a patterned layercomprises: forming a metal oxide layer overlying the dielectric layer onthe semiconductor wafer; and patterning the metal oxide layer into aplurality of metal oxide regions, wherein the metal oxide regions areconfigured to receive nucleic acid macromolecules.
 8. The method of anyof claim 1, wherein forming a patterned layer comprises: forming a metaloxide layer; forming a silicon oxide layer overlying the metal oxidelayer; and patterning the silicon oxide layer, wherein regions of themetal oxide layer not covered by the silicon oxide layer are configuredto receive a nucleic acid macromolecule.
 9. The method of claim 1,further comprising forming a support structure on the semiconductorwafer before attaching the cover wafer to the semiconductor wafer. 10.The method of claim 9, further comprising bonding the cover wafer to thesupport structure.
 11. The method of claim 1, wherein the cover wafercomprises a glass wafer.
 12. The method of claim 1, further comprisingfunctionalizing the sequencing flow cell, wherein functionalizing thesequencing flow cell comprises exposing the flow channel to materialssupplied through the inlet and outlet.
 13. The method of claim 1,wherein singulating the composite wafer structure comprises separatingthe composite wafer structure into individual dies using a wafer cuttingprocess.
 14. A method for forming sequencing flow cells, comprising:providing a semiconductor wafer having a dielectric layer overlying acomplementary metal-oxide-semiconductor (CMOS) layer, wherein the CMOSlayer includes: a photo sensing layer, the photo sensing layer includinga plurality of photodiodes; an electronic circuit layer coupled to thephoto sensing layer for processing sensed signals; and forming apatterned layer on the dielectric layer, the patterned layer havingmetal oxide regions and silicon oxide regions; attaching a glass waferto the semiconductor wafer to form a composite wafer structure, theglass wafer including a plurality of holes and the composite waferstructure including a plurality of sequencing flow cells, wherein eachsequencing flow cell includes: a glass layer having holes configured asan inlet and an outlet of the sequencing flow cell; multiple metal oxideregions and silicon oxide regions; and a flow channel between the glasslayer and the multiple metal oxide regions and silicon oxide regions;and singulating the composite wafer structure to form a plurality ofdies, each die including a sequencing flow cell.
 15. The method of claim14, wherein forming a patterned layer comprises: forming a metal oxidelayer overlying the dielectric layer on the semiconductor wafer; andpatterning the metal oxide layer into a plurality of metal oxideregions, wherein the metal oxide regions are configured to receivenucleic acid macromolecules.
 16. The method of claim 14, wherein forminga patterned layer comprises: forming a metal oxide layer; forming asilicon oxide layer overlying the metal oxide layer; and patterning thesilicon oxide layer, wherein regions of the metal oxide layer notcovered by the silicon oxide layer are configured to receive a nucleicacid macromolecule.
 17. The method of claim 14, 15, or 16, furthercomprising bonding the glass wafer to the semiconductor wafer.
 18. Themethod of claim 14, 15, or 16, further comprising functionalizing thesequencing flow cell, wherein functionalizing the sequencing flow cellcomprises exposing the sequencing flow cell to materials suppliedthrough the inlet and outlet.
 19. A method for forming sequencing flowcells, comprising: providing a semiconductor wafer covered with adielectric layer; forming a patterned layer on the dielectric layer, thepatterned layer having metal oxide regions and oxide regions; forming aplurality of through holes through the semiconductor wafer; attaching aglass wafer to the semiconductor wafer to form a composite waferstructure that includes a plurality of sequencing flow cells, whereineach sequencing flow cell includes: a glass layer; multiple metal oxideregions and oxide regions; and a flow channel between the glass layerand the multiple metal oxide regions and oxide regions, wherein thethrough holes in the semiconductor wafer are configured as inlet andoutlet of the sequencing flow cell; and singulating the composite waferstructure to form a plurality of dies, each die including a sequencingflow cell
 20. The method of claim 19, wherein forming a patterned layercomprises: forming a metal oxide layer overlying the dielectric layer onthe semiconductor wafer; and patterning the metal oxide layer into aplurality of metal oxide regions, wherein the metal oxide regions areconfigured to receive nucleic acid macromolecules.
 21. The method ofclaim 19, wherein forming a patterned layer comprises: forming a metaloxide layer overlying the dielectric layer on the semiconductor wafer;forming a silicon oxide layer overlying the metal oxide layer; andpatterning the silicon oxide layer, wherein regions of the metal oxidelayer not covered by the silicon oxide layer.
 22. The method of claim19, further comprising bonding the glass wafer to the semiconductorwafer.
 23. The method of claim 19, 20, or 21, further comprisingfunctionalizing the sequencing flow cell, wherein functionalizing thesequencing flow cell comprises exposing the sequencing flow cell tomaterials supplied through the inlet and outlet to form hydrophilicsurface regions and hydrophobic surface regions. 24-45. (canceled)